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THE ALKALOIDS Chemistry and Pharmacology VOLUME 43

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THE ALKALOIDS Chemistry and Pharmacology Edited by Geoffrey A. Cordell College of Pharmacy University of Illinois at Chicago Chicago, Illinois

VOLUME 43

Academic Press, Inc. Harcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California92101-4311

United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NWI 7DX Library of Congress Catalog Number:

50-5522

International Standard Book Number:

0- 12-469543-4

PRINTED IN T I E UNITED STA'IES OF AMERICA 9 3 9 4 9 5 9 6 9 1

QW

9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS ......................................................... PREFACE...............................................................

vii ix

Chapter 1 . Allelochemical Properties or the Raison d'Etre of Alkaloids MICHAELWINK I . Introduction ...................................................... I1 . Allelochemical Properties of Alkaloids ............................... 111. Raison d'Etre of Alkaloids ......................................... IV . Conclusions ...................................................... References .......................................................

I 5 86

I03 104

Chapter 2. Mammalian Alkaloids I1 ARNOLDBROSSI 1. Introduction ...................................................... I1 . Mammalian Indole Alkaloids ....................................... 111. Mammalian Isoquinoline Alkaloids .................................. IV . Mammalian Morphine ............................................. V . Alkaloid Formation in Mammals as a Therapeutic Concept ............. VI . Addendum ....................................................... VII . Conclusions ...................................................... References .......................................................

Chapter 3. Amphibian Alkaloids JOHNW . DALY.H . MARTINGARRAFFO.A N D THOMASF. SPANDE I . Introduction ...................................................... I1 . Steroidal Alkaloids ................................................ 111. Bicyclic Alkaloids ................................................. IV . Tricyclic Alkaloids ................................................ V . Monocyclic Alkaloids .............................................. .................................... VI . Pyridine Alkaloids ........ VII . Indole Alkaloids .............................................. VIII . Imidazole Alkaloids ............................................... IX . Morphine ........................................................ X . Guanidinium Alkaloids ............................................. V

186

187 199

242 251 255 257 263 263 264

CONTENTS

vi

XI. Other Alkaloids ................................................... XII. Summary ......................................................... Appendix ........................................................ References .......................................................

CUMULATIVE INDEX OF

INDEX

TITLES ...........................................

.................................................................

269 215 211 28 1

289 291

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ARNOLD BROSSI(1 19), Department of Chemistry, Georgetown University, Washington, D. C. 20057 JOHNW. DALY(189, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 H. MARTINGARRAFFO (185), Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 THOMASF. SPANDE (189, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 MICHAEL WINK (I), Universitat Heidelberg, Institut fur Pharmazeutische Biologie, 6900 Heidelberg, Germany

vii


PREFACE

Over the 43 years since the first volume in this series was published, most of the chapters have provided critical reviews of the many aspects of the chemistry and biology of alkaloids from the plant kingdom. In Volume 43 of “The Alkaloids, Chemistry and Pharmacology” all three chapters adopt a quite different perspective. In Chapter 1 “Allelochemical Properties or the Raison d’Etre of Alkaloids,” Michael Wink examines in considerable detail, and with some healthy speculation, why it is that organisms, such as plants, actually produce alkaloids. Is it, for example, as was previously thought, that alkaloids are the waste products of the organism? Wink says that based on the biological activities observed for many alkaloids and the ecological niche that certain plants occupy, emphatically not. Rather, alkaloids are indeed important compounds to the organism, possibly as antimicrobial or antipredation agents, or as competitive inhibitors for other plants or organisms. The remaining two chapters provide fascinating insights into the alkaloids of mammals and of amphibians. As a follow-up to a chapter published in Volume 21 of this series, Arnold Brossi offers a critical review of the current status of the knowledge of mammalian alkaloids, such as those derived from tryptophan and from phenylalanine, and in particular he reviews the literature regarding the fascinating subject of whether morphine-like alkaloids are indeed mammalian metabolites. Also following up on an earlier review in Volume 21 by Witkop and Gossinger, John Daly, Martin Garraffo, and Thomas Spande present a summary of the diverse groups of biologically active alkaloids that have been isolated and detected from various amphibians. Much of this work, conducted on minute amounts of material, is from the authors’ laboratory and has not been available previously. In summary, this volume maintains the tradition of the series of providing outstanding new reviews of general and specific interest and of updating established areas where there have been significant recent results. Geoffrey A. Cordell University of Illinois at Chicago ix


-CHAPTER1-

ALLELOCHEMICAL PROPERTIES OR THE RAISON D’ETRE OF ALKALOIDS MICHAEL WINK Universitat Heidelberg Institut fur Pharmazeutische Biologie 6900 Heidelberg, Germany

I. Introduction .......................................................................................... 11. Allelochemical Properties of Alkaloids ...................................................... A. Plant-Herbivore Interactions ............................................................... B. Plant-Microbe Interactions ............................................................... C. Antiviral Properties .......................................................................... D. Allelopathic Properties ..................................................................... 111. Raison d’8tre of Alkaloids ..................................................................... A, Concentrations in Plants and Allelochemical Activities.. ......................... B. Presence of Alkaloids at the Right Site and Right Time .......................... C. Importance of Alkaloids for Fitness of Plants ....................................... D. Exceptions to the Rule: Role of Adapted Specialists .............................. IV. Conclusions ........................................................................................ References .........................................................................................

1 .5

8 61

79 82 86 87 89 92 % 103 104

I. Introduction

Plants constitute the major group of photoautotrophic organisms on our planet that are able to use solar energy to fix carbon dioxide into hydrocarbons, such as glucose, and to produce ATP and NADPH, as “fuel” and reduction equivalents, which serve to build up all the other essential components of a cell. Animals and most microorganisms (except the chemo- or photoautotrophic bacteria) are heterotrophic organisms, which rely on complex, plant-made organic molecules for their energy requirement or other metabolic functions. Thus plants serve as a major and ultimate source of food for animals and microorgansims, whether they like it or not. We can safely assume that plants struggle for life and that they have evolved strategies against herbivorous animals or phytopathogenic micro1

THE ALKALOIDS, VOL. 43 Copyright 8 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

MICHAEL WINK

organisms. We must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients. How do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? Because plants do rather well in Nature, this question has often been overlooked. We are well aware of the defensive strategies of higher animals against microbes and predators (1,2,4,15,17,28,494).The complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. Against predating animals, Nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called “poisons”) (1). It is evident that most of these possibilities are not available for plants with their sessile and “passive” life-style. What then is their evolutionary solution? We can distinguish the following defense mechanisms in plants (3,4,7,15,17);the mechanisms are not independent and may act cooperatively and synergistically. We should be aware that many species have additionally evolved specialized traits in this context. 1. Mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). 2. Formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. 3. If plants are wounded or if parts of them are eaten, this is usually not as fatal as the similar situation in animals, since plants can easily replace a lost leaf or branch (so-called open growth). 4. A most important strategy, however, is the production and storage of defense chemicals, which are abundant and a typical trait of all plants. a. Plant surfaces are usually covered by a hydrophobic layer consisting of antibiotic and deterranthepellent cuticular waxes which may contain other biologically active allelochemicals such as flavonoids (3-5,7). b. Cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. Callose and lignin are often accumulated at the site of infection or wounding (6,7)and form a penetration barrier. c. Synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. These proteins are either stored in the vacuole

1. ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

3

or are secreted as exoenzymes into the cell wall or the extracellular space (8,9).These compounds are thus positioned at an “advanced and strategically important defense position.” In addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. As a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced (2-4, 10-21). These allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides,glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~ O U synthesis O may be induced by elicitors (so-called phytoalexins), infection, or herbivory (4,7,22-24). These products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection (25,26)]or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots (2,3,15)]. In animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals (1,4,12,16,17,28-30,494-496,503). In many instances, the animals have obtained the toxins from their host plants (4, 12,15,17,27-33).Hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ‘‘toxins” in this context) in animals is that of defense against predators or microorganisms (1,17,28,494-496). These defense compounds are better known as natural products or secondary metabolites. The latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites (34,35,38). Unfortunately the term “secondary” has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. As discussed in this chapter, just the opposite is true. More than 30,000 natural products have been reported from plants so far (2,4,17).Owing to the sophistication in phytochemical methods, such as chromatography (HPLC, GLC) and spectroscopy (NMR, MS), new products are reported at rapid intervals. Because only 5-10% of all higher plants, which consist of over 300,000 species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large. It is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. Within a taxon secondary metabolites

4

MICHAEL WINK

often share a common distribution pattern and are therefore of some importance for phytochemical systematics. Classic taxonomy, however, has taken little account of alkaloid distribution: If the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. Because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated (233). For many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of Nature (34,35). In 1887 and 1888, Errera and Stahl (92,308,504) published the idea that natural products are used by plants for chemical defense against herbivores. Since the leading plant physiologists of that time were mostly anti-Darwinian, they were not willing to accept the defense argument, which was too much in line with the Darwinian concept. Therefore, this early defense concept was negated and remained forgotten for nearly 60 years. In 1959, Fraenkel(10) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. During the next three decades this concept was improved experimentally, and we can summarize the present situation as follows (2-4,11-223,210). Although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century (34,35). In many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy). In some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the Centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes (15,17). Physiological roles, such as UV protection [by flavonoids or coumarins (4,17)],nitrogen transport or storage (14,36,37),or photosynthesis (carotenoids), may be an additional function. Allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and


5

antimicrobial). Thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). To understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach. It might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. However, we have to understand and accept that chemical defense is not an absolute process. Rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. Plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. During evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them (14,15,32).This is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. The number of these “specialists” is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. We can compare this situation with our immune system: It works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. Nobody would call the immune system and antibodies useless because of these few adapted specialists! We should adopt the same argument when we consider plants’ defenses by secondary metabolites (2). Since secondary metabolites have evolved in Nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. In addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds.

11. Allelochemical Properties of Alkaloids

About 20-30% of higher plants accumulate alkaloids (505,506). The incidence of alkaloid production varies between taxa to some degree; for example, about 60-70% of species of the Solanaceae and Apocynaceae are

6

MICHAEL WINK

alkaloidal, whereas other families contain few alkaloid-producing species. Some alkaloids have a wide distribution in Nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. Alkaloids are not restricted to higher plants (althoughthey are here most numerous); they are also present in club mosses (Lycopodium), horsetails (Equisetum), fungi, and animals such as marine worms (e.g., Nereidae), bryozoans, insects (e.g., Coccinellidae, Solenopsidae), amphibians (toads, frogs, salamanders), and fishes. Alkaloids thus represent one of the largest groups of natural products, with over 10,000 known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan (38-40). In addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates (38-40). Whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. This is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals. Alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as W. 0. James and Kurt Mothes ( 3 4 3 , 505,526)l.Because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. The waste product argument probably came from animal physiology: Carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. A similar situation or need, however, is not applicable for plants. In fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves (2,37,506). If alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. On the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals (16,28,494496). Thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. This idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and Homo sapiens. As a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. (1) The alkaloid should have significant effects against microbes and/or animals in bioassays.

1.

ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

7

(2) The compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. (3) The compound should be present in the plant at the right time and the right place. (4) Evidence should be provided that a particular compound is indeed important for the fitness of a plant. Although more than 10,000 alkaloids are known, only few (-2-5%) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. In most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities. Furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. These objections have to be kept in mind, because an alkaloid is sometimes termed “inactive” in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). In many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. If the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. Another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. However, if a compound is active against Escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. Nevertheless, most of the data obtained in these studies (Tables I-VIII) provide important information which at present permits extrapolation to the function of alkaloids in plants. In this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. In the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail. It is nearly impossible to cover the literature exhaustively. Therefore, an overview of the allelochemical properties of alkaloids is presented. Because of the large amount of data (literature up to 1990 is included), the selection of examples must remain subjective to some degree. Nevertheless, the author would be grateful to receive information or publications about relevant omissions.

8

MICHAEL W I N K

A. PLANT-HERBIVORE INTERACTIONS Because Homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (Tables I through V) and alkaloid-containing plants. Previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products (38,39). In the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates. 1 . Invertebrates

Among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. Entomologists estimate that the number of insects is at least 1 million, but tropical rain forests may harbor up to 20-30 million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists. Most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( I ,4,10,14,15,28-33, 494496,503). Whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. In the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). At the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds. The close association between plants, especially the angiosperms, and insects evolved during the last 200 million years. Some scientists have called this phenomenon a “coevolutionary” process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved (18,505,506).Applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. Times can also be much longer. If plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. For example, Lupinus polyphyllus from North America has a number of specialized herbivores, but is rarely attacked by herbivores in Europe. This lupine left its enemies behind when it was transferred to Europe three centuries ago. About 10 years ago, however, the North American lupine aphid (Macrosiphum albifrons) was introduced to Europe accidentally. This aphid is specialized to alkaloid-rich lupines with lupanine as a major

1.

ALLELOCHEMICAL PROPERTIES OF ALKALOIDS

9

alkaloid. At present, this aphid has spread over most of Europe and is now colonizing its former host, L. polyphyllus (2,503). Insect herbivores can be divided into two large groups whose strategies with respect to the plant’s defense chemistry differ substantially (15). The polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagousinsects are often specialized on one or a small number of (often systematically related) hosts. Polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors (501) that allow the distinction between plants with high or low amounts of “toxins.” The receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates (507). These “generalists,” as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see Table XI). Alternatively, they change host plants rapidly and thus avoid intoxication. In addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products (4,15,17,508). In contrast, mono- and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. For these “specialists” the originally noxious defense compounds are often attractive feeding and oviposition stimulants. These insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes (1,4,10-12,1447,28,31,33,494-496).These observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as Mothes (35)and Robinson (505). However, these specialized insects are exceptions to the general rule. For these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants (32).As compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species. Quite a number of alkaloids have been tested toward herbivorous insects (Table I). In general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>I%, w/w). Given the choice, insects tend to select a diet with no or only a small dose of alkaloids. Also,

TABLE I ACTIVITYOF ALKALOIDS AGAINST HERBIVORES (MOSTLYINSECTSAND OTHERINVERTEBRATES) ED, Alkaloid Alkaloids derived from tryptophan Acetylokaramine Ajmalicine Ajmaline Brucine

-

Cinchonidine

0

Cinchonine Dictamnine Ergocryptine Ergometrine Ergonovine Ergotamine Gramine Harmaline Harman

Effect Insecticidal in Eombyx Feeding deterrent to polyphagous Synfomis (Lepidoptera) larvae Feeding deterrent to polyphagous Synfomis larvae Feeding deterrent to polyphagous Synfomis larvae Feeding deterrent in bees (Apis mellifera) Insecticidal for bees Phagorepellent in Pieris, Eombyx (Lepidoptera) Feeding deterrent to polyphagous Synromis larvae Feeding deterrent in bees Feeding deterrent in Agelaius (Aves) Feeding deterrent in bees Feeding deterrent in Leptinofarsa (Coleoptera) Insecticidal Toxic to Oncopelfus Inhibition of insect spermatophore formation Feeding deterrent to polyphagous Syntornis larvae Toxic to Oncopelrus Feeding deterrent to polyphagous Synfomis larvae Feeding deterrent in aphids Insecticidal for Schiznphis (Aphidoidea) Phototoxicity in larvae of Trichoplusia (Lepidoptera) Feeding deterrent to polyphagous Syntomis larvae Phototoxicity in larvae of Trichoplusia Deterrent to polyphagous larvae

(dml, d g , or %I 10 1% 0.1% 1% 0.05%

0.2%

-

0.1% 0.04% 40 mg/kg 0.007%

-a 1%

0.1% lo00 rng/kg LD, i.p. 40 mg/kg LD, p.0. 1 mg/kg LD, i.p. 206 mg/kg LD, p.0. 100 mg/kg LD, i.p. 152 mg/kg LDlm i.p. 0.34 mg/kg LD, i.v. 19-22 rng/kg, p.0. 178-204 rng/kg LD, i.v. 1.2 mg/kg LDso i.v. 1 . 1 mg/kg LD, i.v. 0.15 mglkg LD, i.v. 62 rng/kg LD, i.v. 80 mg/kg LD, i.v. 3.5 mg/kg LD, i.p. 50 mg/kg LD, i.v. 38 mg/kg LD, i.p. 30-100 mg/kg, p.0. >lo00 mglkg LD, p.0. 4.5 mg/kg LD, i.v. 285 mg/kg LD, i.v. 280 mg/kg LD, i.v. 12.5 mg/kg LD, i.v. 30 mglkg, p.0. 263 mg/kg LD, p.0. 100 mg/kg LD, p.0. 100 mg/kg LD, i.p. 340 mg/kg LD, i.p. 169-184 mg/kg

Ref. 25 7 149 149 149 175 149 258 149 149 149 259 149 149 259 149 149 257 149 149 149 149 149 I 75 I 75 259 259

Strychnine

Toxiferine Vinblastine Vincamine Vincristine Alkaloids derived from phenylalanine and tyrosine Aristolochic acid

VI N

Agelaius

Starling Rat Dog Mouse Mouse Mouse

Mouse

Berberine Bulbocapnine Canadine

Mouse Mouse Mouse

Chelerythrine C helidonine Codeine Colchiceine Colchicine

Galanthamine

Mouse Mouse Mouse Mouse Mouse Rat Man Agelaius Starling Mouse Rat Mouse Mouse

Glaucine

Mouse

Corydaline Emetine

LD, LD, LD, LD,, LD,, LD, LD, LD,

p.0. 6 mg/kg

i75

p.0. 6 mg/kg 175 i.v. 0.9 mg/kg 149 p.0. 0.3-1.2 mg/kg, S.C. 0.003-0.02 mg/kg 259 i.p. 0.03 mg/kg 258 i.v. 9.5 mglkg 149 i.v. 75 mglkg, p.0. lo00 mg/kg 149 i.p. 5.2 mg/kg 149

LD, i.v. 38(m)-70(f) mg/kg, p.0. 56(m)-106(f) mg/kg LD5o i.p. 23 mg/kg LD, p.0. 413 mg/kg LDm p.0. 940 mg/kg, S.C. 790 mglkg, i.v. 100 mg/kg LDlmS.C. 95 mg/kg LD, i.v. 35 mg/kg LD, S.C. 300 mglkg LD5o i.p. 84 mg/kg LD, i.v. 4.1 mg/kg LD, i.v. 1.6 mglkg LDlWp.0. 0.1-0.3 mg/kg LD, p.0. 32 mglkg LD, p.0. 21 mglkg LD, i.v. 135 mg/kg LD, i.v. 12.1 mg/kg LD, S.C. 32 mg/kg LD50 i.v. 8 mg/kg, p.0. 18.7 mg/kg, S.C. 1 1 . 1 mg/kg LDS0i.v. 98 mg/kg, p.0. 401 mg/kg

149 149 259 149 259 149 149 149 149 149 259 175 17.5 149 149 149 149 149

(continued)

TABLE I1 (Continued) Alkaloid

I sot hebaine Mescaline Morphine Nuciferine Papaverine Protopine Sanguinarine oI N

Tazettine Tetrahydropalmatine Thebaine

Tubocurarine Steroidal alkaloids Batrachotoxin (frog) Batrachotoxinin A Chaconine Germerine Jervine Protoveratrine Rubijervine

Test System

LD

Mouse Ratlmouse Mouse Rat Mouse Mouse Rat Mouse Mouse Mouse Mouse Frog Rabbit Rabbit Mouse Rat

LD, LD, LD, LD, LD, LD, LD,, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD,

Mouse Man Mouse Rat Rat Mouse Rabbit Rat

LD, S.C. 2 /&kg Lethal dose 200 pg LD, S.C. 1 mg/kg LD, i.p. 84 mg/kg LD, S.C. 3.7 mglkg LD, i.v. 9.3 rng/kg Lethal dose 0.1 mglkg LD, i.v. 70 mg/kg

Mouse Agelaius

i.p. 26 mg/kg p.0. 100 mg/kg i.v. 226-318 mg/kg p.0. 240-280 mg/kg i.v. 27.5 mg/kg, S.C. 150 mg/kg i.v. 20 mg/kg, S.C. 370 mg/kg 100 mg/kg i.p. 36-102 mg/kg i.v. 29 mg/kg, p.0. 1658 mg/kg S.C. 102 mg/kg, i.v. 16 mg/kg i.v. 100 mg/kg, i.p. 420 mg/kg i.p. 111 mg/kg i.p. 20 mg/kg i.p. 50 mg/kg i.p. 3-4 mglkg S.C. 14 mglkg p.0. 33.2 mg/kg p.0. 21.8 mg/kg

Ref. 260 I 75 149 260 149 149 259 260 149 149 259 260 259 260 260 149 149 149 149 259 149 259 259 149 259 149

Samandarine Solanine

Tomatidine Tomatine Veratridine Tropane alkaloids Apoatropine Atropine Cocaine -4 N

Pyrrolizidine alkaloids 7-Angeloylheliotridine Echimidine Echinatine Europine Heliotrine Heliotrine N-oxide Jacobine Lasiocarpine Monocrotaline Retronecine Retrosine Retrorsine N-oxide

Frog Mouse Rabbit Hens’ eggs Monkey Rat Mouse Rabbit Agelaius Rat Mouse

LD,, 19 mglkg LDIM3.4 mglkg LD,, 1 mg/kg LD,, 0.3-1.5 mg/egg L D I Mi.p. 40 LD, i.p. 67 mg/kg, p.0. 590 mg/kg LDN i.p. 42 mg/kg Lethal dose 20-30 mg/kg i.p. LDN p.0. 100 mg/kg LD, p.0. 900-1OOO mg/kg LD, i.p. 1.4 mg/kg

263 263 263 26 I 262 262 259 259 I 75 149 149

Mouse Rat Man Rat Man

LD, p.0. 160 mg/kg, i.p. 14.1 mg/kg LD, p.0. 750 mg/kg Paralytic dose >I0 mg LD, i.v. 17.5 mglkg Lethal dose >30 mg i.v.

149 149 259 149 259

Rat Rat Rat Rat Rat Rat Rat Rat Rat Mouse Rat Rat

LD, i.p. 260 mg/kg LD, i.p. 200 mg/kg LD, i.p. 350 mglkg LD, p.0. 1OOO mglkg LD, i.p. 300 mg/kg LD, i.p. 2500(f)-5OOO(m) mg/kg LDSoi.p. 138 mg/kg LD, i.p. 260 mglkg LDN i.p. 175 mg/kg, p.0. 71 mglkg LD, i.v. 634 mg/kg LD, i.p. 30-150 mg/kg LD, p.0. 250 mg/kg, i.p. 48 mg/kg

259 259 259 264 259 259 259 259 149,259,265 149 265 265 (continued)

TABLE I1 (Continued) Alkaloid Senecionine Seneciph ylline Supinine Quinolizidine alkaloids Cytisine

Test System Rat Mouse Rat Rat

LD, LD, LD, LD,

Cat Dog Goat Mouse

LDlw S.C. 3 mg/kg LD,, S.C. 4 mg/kg LD,, S.C. 109 mg/kg LD, i.v. 1.7 mg/kg, i.p. 9.3 mg/kg, p.0. 101 mg/kg LD, i.p. 200-400 mg/kg LD,, i.p. 228 mg/kg, S.C. 456 mg/kg LD, i.p. 199 mg/kg LD, i.p. 172 mg/kg LD,, i.p. 28-30 mglkg LD,, i.p. 22-25 mg/kg LD, i.p. 80 mg/kg LD, i.p. 180-192 mg/kg LD, i.p. 210 mg/kg LD,, i.p. 175 mg/kg, p.0. 410 mg/kg LD5, i.p. 150 mg/kg LD, i.p. 750 mg/kg, i.v. I50 mg/kg LD, i.v. 21 mg/kg, i.p. 51 mg/kg LD, i.v. 29 mg/kg LD, i.p. 177 mg/kg, p.0. 1464 mg/kg LD, i.p. 690 mg/kg

m N

Ep iIup in ine 13-H ydroxylupanine

Lupinine Lupanine

Matrine Matrine N-oxide N-Methylycytisine Nupharidine 17-Oxolupanine

LD

Rat Guinea Rat Mouse Guinea Guinea Mouse Rat Guinea Mouse Mouse Mouse Mouse Mouse Rat Mouse

pig pig pig

pig

50 mg/kg, i.p. 85 mg/kg i.v. 64 mg/kg i.p. 77 mg/kg i.p. 450 mg/kg

Ref. 259 149 259 259 278 278 278 149 275 268 2 75 2 76 268 268 273 273 273 274 31 I 31 I 149 259 275 277

Sparteine

Guinea pig Rat Mouse Rabbit Rabbit Dog Pigeon

Miscellaneous alkaloids Aconitine

Actinobolin t 4 W

Adenine a-Amanitin P-Amanitin Anabaseine Antimycin A Arecoline Benzoylaconitine 2,3’-Bipyridyl Caffeine

Calcimycin

Mouse Rat Cat Man Mouse Rat Rat Mouse Mouse Mouse Mouse Mouse Dog Rat Mouse Agelaius Mouse Hamster Rabbit Rat Mouse

LDIMi.p. 23-30 mglkg LD, i.p. 42-44 mglkg, S.C. 68-75 mg/kg LD, i.p. 55(m)-67(f) mg/kg, i.v. 17(m)-20(f) mglkg, p.0. 350(m)-510(f) mglkg LD,, p.0. 450 mg/kg Lethal dose i.v. 20-30 mglkg Lethal dose i.v. 50-70 mglkg Lethal dose i.v. 40-50 mglkg LD, i.v. 0.166 mg/kg, i.p. 0.328 mg/kg, p.0. - 1 mg/kg LD, i.v. 0.08-0.14 mglkg LD, i.v. 0.07-0.13 mg/kg Lethal dose p.0. 1.5-5 mg LD, i.v. 800 mglkg LD, i.v. I550 mglkg LD, p.0. 745 mg/kg LD, i.p. 0.1 mg/kg LD, i.p. 0.4 mg/kg LD, i.v. 84 pg/kg LD, i.p. 1.8 mg/kg, S.C. 1.6 mg/kg LD, S.C. 100 mglkg LD, S.C. 5 mg/kg LD, i.v. 27 mg/kg LDWi.v. 3500 pg/kg LD, i.p. 316 mglkg LD, p.0. 127(m)-137(f) mglkg LD, p.0. 230(m)-249(f) mg/kg LD, p.0. 246(m)-224(f) mg/kg LD, p.0. 200 mg/kg LDw .. i.p. . 10 mg/kg - -

268

269 270 271 272 272 272 149 259 259 259 149 149 149 149 149 230 149 149 149 259 230 149 149 149 149 259 149 (continued)


Test System

LD

Ref. ____

s

Carubicin Carzinophilin Coniine Cycloheximide Damascenine Daunorubicin Delphinine

Epinephrine (adrenaline) Glomerine H ypaconitine Lappaconitine Lycoctonine Maitotoxin (algaelfish) Maytansine Mesaconitine

Mouse Mouse Agelaius

Guinea pig Mouse Mouse Mouse Frog Rabbit Mouse Mouse Mouse Mouse Mouse Mouse Rat Mouse

LD, LD, LD, LD,, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD, LD,, LD, LD,

p.0. 7.3 mglkg, i.v. 1.3 mglkg i.v. 150 pglkg p.0. 56 mglkg p.0. 150 mglkg, S.C. 40 mg/kg i.v. 150 mg/kg p.0. 1800 mglkg i.v. 26 mg/kg i.p. 0.05-0.1 mglkg i.p. 1.5-3.0 mg/kg i.p. 4 mglkg p.0. 17-34 mg/kg S.C. 1.2 mglkg i.v. 6.9 mg/kg, p.0. 20 mglkg i.p. 350 mglkg i.p. 0.17 pg/kg S.C. 0.48 mg/kg S.C. 0.2 mglkg

149 149 175 259 149 149 149 267 267 149 259 259 149 267 259 149 259

Methyl-lycaconitine Mitomycin Muscimol Nemertilline Nereistoxin Nicotine

Nornicotine

W

Frog Mouse Mouse Rat Mouse Mouse Agelaius Starling Mouse

Ochratoxin Palytoxin Pellertierine Ricinine Saxitoxin

Rat Rabbit Rat Mouse Rabbit Agelaius Mouse

Tetrodotoxin

Guinea pig Mouse

Theobromine

Rat

LD, i.p. 3.0-3.5 mglkg LD, i.p. 18 mglkg LD, i.v. 5-10 mglkg LD, p.0. 45 mg/kg LD, i.v. 500 pg/kg LDlw S.C. 38 &kg LD, p.0. 17.8 mglkg LD, p.0. 42 mg/kg LD, i.v. 0.3 mglkg, i.p. 9.5 mglkg, p.0. 230 mg/kg LD, i.p. 23.5 mglkg LD, i.v. 3 mg/kg LD, p.0. 20-22 mglkg LD, i.v. 0.45 pglkg, i.p. 0.05-0.15 pg/kg LD, i.v. 40 mglkg LD, p.0. 42 mg/kg LD, i.p. 10 p g / k g , i.v. 3.4 mg/kg, p.0. 263 mglkg LD, P.O. 135 pglkg LD, i.p. 10 pglkg, S.C. 8 p g / k g , p.0. 0.3 mg/kg LD, p.0. 950 mg/kg

267 267 149 266 230

221 175 175 149 149 149 149 149 149 259 149 259 149,259 259

TABLE I11 CYTOTOXIC ACTIVITY OF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Annomontine Apparicine Bisnordihydrotoxiferine Boldine Brevicolline Camptothecine

W h)

Canthin-Gone Cinchonidine Cinchonine Conoduramine Conodurine Coronoaridine Ellipticine 1GEpi-(Z)-isositsirikine 9-Epivoacarine Gabunamine Gabunine Harmaline Harman Harmine Harmol 20-H ydrox yvoacamidine

Isovoacangine Leurosidine Methoxyannomontine

Effect Antiamebic Cytotoxic to P388 cells Inhibition of sarcoma 180 Inhibition of human epidermoid carcinoma of larynx Photogenotoxic in CHO cells Antitumor properties, L1210 Walker sarcoma Cytotoxic to KB and P388 cells Photogenotoxic in CHO cells Growth inhibition of Plasmodium falciparum Growth inhibition of Plasmodium falciparum Inhibition of P388 leukemia cells Inhibition of P388 leukemia cells Cytotoxic to P388 cells Antitumor agent in L1210 cells Antineoplastic to KB and P388 cells Cytotoxic to P388 cells Inhibition of P388 leukemia cells Inhibition of P388 leukemia cells Growth inhibition of Trypanosoma cruzi Photogenotoxic to CHO cells Growth inhibition of Trypanosoma cruzi Growth inhibition of Trypanosoma cruzi Growth inhibition of Trypanosoma cruzi Antineoplastic Inhibition of P388 leukemia cells Antitumor activity Antiamebic

ED54 50 pg/ml

18 mg/ kg 0.17-0.53 pg/ml

200 ng/ml 27-130 ng/ml 20 pg/ml 26 pg/ml 0.43pglml

-

1.2 p g h l I .7 pg/ml 1.3 pg/ml 3.2 pg/ml

18 pglrnl

Ref. 257 283 284 285 57 286 283 57 287 287 281 281 283 288 280 281 281 281 289 57 289 289 289 282 281 282

2v

Antitumor activity in L1210, P388 Inhibition of Eagle carcinoma of nasopharynx 9-Methox yellipticine Cytotoxic Growth inhibition of Trypanosoma cruzi and Crirhidia Olivacine Tumor inhibition in L1210 cells Cytotoxic to KB cells Inhibition of P388 leukemia cells Pericyclivine Inhibition of P388 leukemia cells Perivine Relefolonium Inhibition of animal/human cells Growth inhibition of Plasmodium falciparum Quinidine Growth inhibition of Plasmodium berghei Quinine Growth inhibition of Trypanosoma cruzi Growth inhibition of Plasmodium falciparum Cytotoxic to Walker 256 carcinosarcoma Reserpine Inhibition of P388 leukemia cells Tabernamine Cytotoxic to P388 cells Tubotaiwine N4-oxide Tubulosine Inhibition of leukemia and carcinoma cells Amebicidal Cytotoxic to KB and P388 cells Vallesiachotamine Growth inhibition of Trypanosoma cruzi Vinblastine Antitumor activity in Hodgkin’s disease, testicular cancer Antitumor activity in childhood leukemia, Wilm’s Vincristine tumour, lymphomas Vinleurosine Antitumor activity Vinrosidine Antitumor activity Voacamine Cytotoxic to P388 cells Alkaloids derived from phenylalanine/tyrosine Antioquine Growth inhibition of Leishmania Aristolochic acid Antitumor activity Cytotoxic to KB cells Armepavine N-oxide 9-Methox ycamptothecine

1-Methoxycanthin-Gone

W

0.4 pg/ml 13 pg/ml 20 pg/ml 10 pM

22-80 nglml 50 mg/kg

-

45-280 ng/ml

-

2.1 pg/ml 1.8 pg/ml 0.01-0.oooO1 pg/ml

-

1.1-3.5 pg/ml

283 279 282 290 291 292 281 281 256 287 293 289 287 282 281 281 174 304 280 289 286 286

2.6 pg/ml

282 282 281 294 282 295 (continued)


Effect

Ref.

~

Berbamine Berberine

W P

Berbermbine Capnoidine Chelerythrine Chelidonine Chondrodendrine Cissamparein Claviculine Cocsuline Colchicine Coptisine Coralyne Corpaine Corydine Curin Cycleacurine Cycleadrine Cycleanine Cycleanorine Cycleapeltine Daphnandrine Dehydroemetine Demecolcine Dicentrine N-oxide

Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Inhibition of Plasmodium fakiparum Cytotoxic properties Antitumoral Growth inhibition of Trypanosoma brucei Antitumor activity Cytotoxic Growth inhibition of Leishmania Active against nasopharyngal carcinoma Growth inhibition of Plasmodium berghei Growth inhibition of Leishmania Cytotoxic activities Cytotoxic activity Antileukemic to L1210, P388 cells Antitumor Growth inhibition of Trypanosoma brucei Cytotoxic activity Active against nasopharyngal carcinoma Cancerostatic Cancerostatic Growth inhibition of Leishmnnia Cancerostatic Cancerostatic Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Low anticancer activity Cytotoxic activity Cytotoxic to KB cells

294 289 2% 282 297 293 298 282 299 282 293 299 282 297 300 297 301 282 282 282 282 299 282 282 299 299 302 282 295

Emetine Fagaronine Fangchinoline Glaziovine Gyrocarpine Isochondrodendrine Isocorypalmine Jatrorrhizine Krukovine Limacine Liriodenine Lycorine 0-Methylatheroline Nitidine Obaberine Oxodicentrine Oxoglaucine 0x0-0-methylbulbocapnine Oxopurpureine Oxoxylopine Palmatine Penduline Pheantine Protopine Pseudo1ycorine

Growth inhibition of Trypanosoma cruzi Weak anticancer activity Cytotoxic to KB cells, leukemia L1210, P388 cells Active against nasopharyngal carcinoma Cytotoxic Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Active against nasopharyngal carcinoma Antitumoral Inhibition of Plasmodium falciparum Growth inhibition of Leishmania Growth inhibition of Leishmania Active against nasopharyngal tumors Cytotoxic to A-549, HCT-8, KB, P388 cells Toxic to Rauscher virus NIH13T3 cells Cytotoxic Antileukemic to mouse, L1210, P388 cells Antitrypanosomal Growth inhibition of Leishmania Growth inhibition of Trypanosoma cruzi Cytotoxic to A-549, HCT-8, P388 cells Cytotoxic to HCT-8, KB cells Cytotoxic to A-549, HCT-8 cells Cytotoxic Cytotoxic to A-549, HCT-8, KB, P388 cells Antitumoral Inhibition of Plasmodium falciparum Cytostatic Growth inhibition of Leishmania Cytotoxic Toxic to Rauscher virus NIH/3T3 cells

289 303 88,286 282 282 299 294 282 297 2% 299 299 282 295 147 282 300

-

1.O pglml

299 294 295 295 295 282 295 297 2% 282 299 282 147 (continued)

TABLE 111 (Continued) Alkaloid Sanguinarine Tetrandrine Thalfoeditine Thalicarpine (=thaliblastine) Thalidasine Xylopine Acndone alkaloids Acronycine Atalaphillidine Atalaphillinine W

QI

Citpressine I Citracidone I Citrusinine I Dercitine (sponge)

Des-N-methylnoracronycine Dimethoxyacronycine Glandisine Glycobismine A Glycocitrine I Glyfoline Grandisine

5-Hydroxy-N-methylseverifoline 5-H ydroxynoracronycine

Effect

ED%

Ref.

Antitumor activity Active against Walker carcinoma cells Active against carcinoma 256 in rats Antileukemic to Walker S, TLX-5 cells Active against carcinosarcoma 256 in rats Cytotoxic to A-549, HCT-8, KB, P388 cells

298 286,305 282 306 282 295

Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Active against P388, HCT-8 cells Growth inhibition of Plasmodium yoelii Active against some leukemia L1210 cells Growth inhibition of Plasmodium yoelii Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelli Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii Active against mouse leukemia L1210 cells Active against mouse leukemia L1210 cells Growth inhibition of Plasmodium yoelii

145 307 145 307 145 307 145 145 145 144 307 145 307 307 145 307 I45 I45 307 145 145 307

10 Fg/ml 10 pglml 10 pg/ml

-

10 pg/ml 10 pg/ml

10 pg/ml

Melicopine 5-Methox yacronycine N-Methylatalaphilline

1.3-0-Methyl-N-methylacridone Normelicopidine Steroidal alkaloids Solamargine &Solamarine Solasodine Solasoninelsolamargine Pyrrolizidine alkaloids Echinatine-N-oxide Europine N-oxide Fulvine Heliotrine Heliotrine N-oxide Indicine N-oxide Lasiocarpine Monocrotaline Senecionine Senecionine N-oxide Spectabiline Supinine Quinolizidine alkaloids Matrine Oxymatrine Miscellaneous alkaloids Arecoline

Antitumor activity Active against mouse leukemia LIZ10 cells Growth inhibition of Plasmodium yorlii Active against mouse leukemia L1210 cells Growth inhibition of Plasmxfirrm yorlii Growth inhibition of Plasniodirtm yoelii Antitumor activity

282 145

Cytotoxic to PLC. PRF cells Antitumor activity Cytotoxic to PLC, PRF cells Inhibition of skin cancer

310

Active against P388 mouse leukemia Active against P388 mouse leukemia Antitumor activity Antitumor activity Antitumor activity Active against P388 mouse leukemia Antitumor activity Antileukemic effects Antitumor activity Antitumor activity Antitumor activity Antitumor activity

311 31 I

31 I

Antitumor activity in Ehrlich ascites tumor Antitumor activity in mouse sarcoma 180 Antitumor activity in mouse sarcoma 180

31 I 31 I 311

Growth inhibition of Tryprrnosornu crrczi .. Inhibition of intestinal cestodes and nematodes

312

145 145

307 307 282

282

310 309

282 282 282 282 286 282 282 282 282

289

(continued)

TABLE I11 (Continued) W

00

Alkaloid Aristolactam Atropine Cephalomannine Crinamine Cryptopleurine Demethyltylophorinine Deoxyhaningtonine Didemnins trans-Dihydronarciclasine Diplamine Ecteinascidins (tunicate) Emarginatine B Febrifugine Haemanthamine Haningtonine

Effect Antitumoral in lung cells, colon tumors Growth inhibition of Trypanosoma cruzi Antileukemic agent Active against KB cells Toxic to Rauscher virus NIHl3T3 cells Active against KB carcinoma cells Antitumor activity Active against lymphocytic leukemia Antitumor activity in L1210 cells Active against P388 mouse leukemia Cytotoxic toward L1210 leukemia cells Active in P388 mouse leukemia, L1210 cells Cytotoxic in KB cells Antitumor activity Toxic to Rauscher virus NIHl3T3 cells Active against lymphocytic leukemia

EDs

Ref.

-

313 289 314 315 147 133 282 316.317 109 323 189 109 318 282 147 316,317

-

0.38 pglml 0.2 pglml

0.01-0.005 pglml 0.003 pglml 0.002 pglml 0.0001-0.08 pglml 0.4 pglml

-

0.2 pglml

-

'

Homohaningtonine 6-Hydrox ycrinamine

Isohaningtonine Jatropham Maytansine Narciclasine Odorinol F’ancratistatin Patellamid A (tunicate) Pilocarpine F’recriwelline Pretazettine

W

Sesbanimide Solapalmitenine Solapalmitine Tylocrepine Tylophorine Ungeremine

Active against lymphocytic leukemia Toxic to Rauscher virus NIH/3T3 cells Active against lymphocytic leukemia Active in P388 mouse leukemia Antileukemic agent Toxic to Rauscher virus NIH/3T3 cells Antileukemic agent Antineoplastic Antileukemic agent Antitumor activity Toxic to Rauscher virus NIH/3T3 cells Antileukemic agent Toxic to Rauscher virus NIH/3T3 cells Antileukemic agent Antitumor activity Antitumor activity Antitumor activity Antitumor activity Cytotoxic to S180 tumor cells

0.2 pg/ml 0.005 pg/ml

-

2-4 pglml

-

0.05 pg/ml

0.05 pg/ml

-

316,317 147 316,317 282 319 147 320 321 320 282 147 322 147 320 282 282 282 282 114

TABLE IV MOLECULAR TARGETS OF ALKALOIDS: PROTEINS, NUCLEIC ACIDS,BIOMEMBRANES, A N D ELECTRON CHAINS Alkaloid Indole and quinoline alkaloids Acronycine Anonaine BoIdine Brucine

Camptothecine P-Carboline-1-propionicacid Dictamnine Ellipticine

g Ergot alkaloids Ervatamine Eseridine Eserine (physostigmine) 1-Ethyl-P-carboline Gelsemine Gramine Harmaline Harman Harmine Harmol Isoboldine

Effect

Ref.

Inhibition of nucleoside transport Inhibition of adenylate cyclase Quenching of singlet oxygen Quenching of singlet oxygen Inhibition of muscle lactate dehydrogenase Binding to glycine receptor Inhibition of 45 S rRNA transcription Inhibition of cAMP phosphodiesterase Monofunctional photoaddition to DNA Intercalation with DNA Inhibition of mitochondria1 respiration Inhibition of cytochrome c oxidase, interaction with phospholipids Interaction with dopamine, serotonin, and norepinephrine receptors Inhibition of Na+ channels Cholinergic Inhibition of acetylcholinesterase Inhibition of cAMP phosphodiesterase Modulation of glycine neurochemical activity Uncoupling of photophosphorylation Inhibition of Na+,K+-ATPase, Na+ transport, and monoamine oxidase A Interaction with insect synapses Binding to DNA Inhibition of monoamine oxidase Interaction with insect synapses Binding to DNA Interaction with insect synapses Inhibition of adenylate cyclase

360 361 362 362 363 364 365,366 357 367 368 369 358 370,371 372 149 259,373 357 364 374 375,376 377 166 376 377 378 377 36 I

Melinone F 9-Methoxyellipticine Norharman Normelinone F Pseudanel pseudene Quinine

Reserpine Serotonin Skimmianine Strychnine

P Vincristine Tetrahydro-/3-carboline Toxiferine Tryptamine Tubocurarine Vinblastine Vincamine Yohimbine

Binding to DNA Inhibition of cytochrome c oxidase, interaction with phospholipids DNA intercalation Binding to DNA Binding to DNA Inhibition of mitochondria1 electron transport Intercalation with DNA Modulation of ion channels Inhibition of glucose response in chemosensory cells Quenching of singlet oxygen Inhibition of noradrenaline transport Interaction with endogenous neurotransmitter, inhibition of pyridoxal kinase, aromatic amino acid decarboxylase, histamine methyltransferase Intercalation in DNA, photoaddition Binding to glycine receptor Quenching of singlet oxygen Inhibition of muscle lactate dehydrogenase Binding and dimerization of tubulin Inhibition of protein biosynthesis and DNA-dependent RNA polymerase Inhibition of intracellular transport Inhibition of biogenic amine uptake Inhibition of monoamine oxidase Binding to acetylcholine receptor Inhibition of pyridoxal kinase, tyrosine-tRNA ligase Binding to acetylcholine receptor Binding and dimerization of tubulin Inhibition of protein biosynthesis and DNA-dependent RNA polymerase Inhibition of intracellular transport Quenching of singlet oxygen Adrenergic blocking agent

379 358 359 166 379 380 381 382 383 362 312 221,376 57 364 362 363 384-386 387 388 389 389 390 376 391 384-386 387 388 36 1 312 (continued)

TABLE IV (Continued) Alkaloid Alkaloids derived from phenylalanine/tyrosine Alpinigenin Avicine Berbamine Berberine

N P

Bicuculline Bulbocapnine Canadine Cepharanthine Chelerythrine Chelidonine Chelilutine Colchicine

Columbamine Coptisine

Effect

Ref.

Inhibition of mitochondria1 respiratory chain Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Interaction with plasma membranes Inhibition of reverse transcriptase Intercalation with DNA Inhibition of aldose reductase Inhibition of acetylcholinesterase, alcohol dehydrogenase, aldehyde reductase, diamine oxidase, tyrosine decarboxylase, RNA synthesis Modulation of GABA neurochemical activity Inhibition of peripheral dopamine receptors Inhibition of aldose reductase Interaction with plasma membranes Intercalation with DNA Inhibition of reverse transcriptase, alanine and aspartate aminotransferases Inhibition of reverse transcriptase Inhibition of microsomal monooxygenase Inhibition of DNA polymerase Depolarization of microtubules, inhibition of urate-ribonucleotide phosphorylase Binding to tubulin, inhibition of microtubule polymerization Inhibition of intracellular transport Inhibition of RNA synthesis Inhibition of butrylcholinesterase Intercalation with DNA Inhibition of acetylcholinesterase, alcohol dehydrogenase

392 393 393 394 395 3%-398 399 297 364 149 399 394 400 259,401 401 402 393 376,441,442 384,448 388 12 297 3% 297

Coralyne

Corlumine Corysamine Demethylpapaverine Dihydrochelerythrine Dihydrosanguinarine Domesticine Emetine Ephedrine Fagaronine P W

Galanthamine Glaucine Isoboldine Jatrorrhizine Laudanosine 0-Methylfagaronine 13-Methylpalmatine Nandazurine Nantenine Nitidine

Nuciferine

Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Inhibition of catechol 0-methyltransferase, alcohol dehydrogenase Inhibition of acetylcholinesterase, RNA polymerase, tRNA methyltransferase Modulation of a-aminobutryric acid (GABA) neurochemical activity Inhibition of alcohol dehydrogenase Inhibition of aldose reductase Inhibition of reverse transcriptase Inhibition of reverse transcriptase Inhibition of aldose reductase Inhibition of protein biosynthesis Modulation of noradrenaline release and noradrenaline receptors Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Inhibition of acetylcholinesterase Quenching of singlet oxygen Inhibition of aldose reductase Inhibition of butyrylcholinesterase Modulation of glycine neurochemical activity Inhibition of reverse transcriptase Inhibition of reverse transcriptase Inhibition of aldose reductase Inhibition of aldose reductase Intercalation with DNA Inhibition of reverse transcriptase, DNA polymerase Inhibition of tRNA methyltransferase Inhibition of Na+, K+-ATPase Blocking of receptors for neurotransmitters (glutamate, aspartate, acetylcholine)

386 403 298 297 364 297 399 401 401 399 404 12,312 88,400 403,404 405 361 399 297 364 403 297 399 399 400 403 298 298 260 (continued)

TABLE IV (Continued) Alkaloid Palmatine Papaverine

Salsolinol Sanguinarine

Stepholidine Tetrah ydroberberine Tetrah ydroisoquinoline Tetrahydropalmatine Tetrandrine Thebaine Tubulosine Tyramine Polyhydroxy alkaloids Alexine

Effect

Ref.

Inhibition of reverse transcriptase Inhibition of aldose reductase Inhibition of acetylcholinesterase Inhibition of aldose reductase Inhibition of GABA response in chemosensory cells Inhibition of glucose response in chemosensory cells Inhibition of phosphodiesterase Inhibition of monoamine oxidase Inhibition of biogenic amine uptake Uncoupler of respiration and oxidative phosphorylation in mitochondria Inhibition of photosynthetic phosphorylation Inhibition of reverse transcriptase Inhibition of Na+, K+-ATPase Intercalation with DNA Inhibition of catecholamine uptake Inhibition of adenylate cyclase Inhibition of catechol 0-methyltransferase Inhibition of uptake of biogenic amines Inhibition of catecholamine uptake Inhibition of respiratory chain in mitochondria Inhibition of aldose reductase Interaction with plasma membrane Inhibition of acetylcholinesterase Inhibition of protein biosynthesis nhibition of tyrosine-tRNA ligase odulation of noradrenaline release

395 399 297 297 383 383 406 389 389 143 407 401 259,408 400,409 297 297 389 389 297 392 399 243 260 404 3 76 I2

Inhibition of myrosinase/glucosinate hydrolysis at 64-860 pM

212,410,4/1

L

Castanospermine

Deoxynojirimycin I -Deoxynojirimycin 1J-Dideox y- I ,5-imino-D-mannitol

2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine 6-Epicastanospermine Homonojirimycin

2

Nojirimycin Swainsonine Purine alkaloids Caffeine Theophylline Quinolizidine alkaloids Angustifoline Cytisine 13-Hydroxylupanine Lupanine Matrine

Inhibition of glucosidases Inhibition of myrosinase Inhibition of insect disaccharidases Inhibition of myrosinase/glucosinate hydrolysis Inhibition of glucosidase Inhibition of myrosinase/glucosinate hydrolysis Inhibition of a-mannosidase, trehalase Inhibition of myrosinaselglucosinate hydrolysis Inhibition of glucosidase Inhibition of trehalase, invertase Inhibition of a-glucosidase Inhibition of myrosinase/glucosinate hydrolysis Inhibition of glucosidase Inhibition of a-amylase, P-fructofuranosidase, a-glucosidase Inhibition of a-mannosidase, mannosidase I1

150 412 197 212,410,41I 150,212 212,410 212,410 212,410 150,212 212 411 212,410,411 413 376 376,414

Inhibition of cAMP phosphodiesterase, dATP(dGTP)-DNA purinetransferase Inhibition of cAMP phosphodiesterase

202,376

Inhibition of Phe-tRNA binding to ribosomes Inhibition of Phe-tRNA binding and elongation Inhibition of Phe-tRNA binding Inhibition of in uitro translation (wheat germ) Inhibition of Phe-tRNA binding to ribosomes Inhibition of in uitro translation (wheat germ) Inhibition of Phe-tRNA binding and elongation Inhibition of Phe-tRNA binding to ribosomes Inhibition of Phe-tRNA binding and elongation Inhibition of in uitro translation (wheat germ) Inhibition of neural glutamate action

417 99,422 56 56 417 56 99,422 41 7 99,422 56 420

202,415

(continued)

TABLE IV (Continued) Alkaloid 17-Oxosparteine Sparteine

13-Tigloylox ylupanine

Effect

Ref.

Inhibition of Phe-tRNA binding Inhibition of in uitro translation (wheat germ) Modulation of Kt channels Inhibition of Phe-tRNA binding to ribosomes Inhibition of GABA response in chemosensory cells Increase in insulin release in /3 cells Inhibition of aminoacyl-tRNA synthase Inhibition of Phe-tRNA binding and elongation Inhibition of in uitro translation (wheat germ) Inhibition of Phe-tRNA binding Inhibition of in uitro translation (wheat germ)

56 56 416,418 417 383 419 421 99,422 56 56 56

Alkylation of DNA and proteins Inhibition of acetylcholinesterase Modulation of pulmonary Naf/K+ pumps

425,426 424 423

Activation of Nat channels Depolarizes membranes Disruption of biomembranes by cholesterol binding Inhibition of acetylcholinesterase Inhibition of acetylcholinesterase Inhibition of acetylcholinesterase Blocking of action potential Blocking of action potential Inhibition of inactivation of Nat channels, depolarization of membranes Inhibition of cholesterol biosynthesis Disruption of biomembranes Binding of cholesterol, hemolysis Inhibition of acetylcholinesterase

427,428 234,429 430,433 431,432 432 432 234 234 234,259 434 435 435 431

Pyrrolizidine alkaloids 01 4

2.3-Deh ydropyrrolizidines

Heliotrine Monocrotaline Steroidal alkaloids Batrachotoxin (frog) Cevadine Chaconine Commersonine Demissine Isorubijervine Muldamine Protoveratrines A,B Solacongestidine Solamargine

Solanine Solanidine Solasonine Tomatine Veratramine Veratridine Tropane alkaloids Atropine

3

Cocaine Miscellaneous alkaloids Aconitine Amanitin Anabaseine Arecoline Batrachotoxin Capsaicine Cassaine Cryptopleurine Cycasin (=methylazoxymethanoI) Dendrobine DIMBOA/MBOA

Complexing with sterols, membrane disruption Inhibition of acetylcholinesterase Inhibition of GABA response in chemosensory cells Inhibition of acetylcholinesterase Synergistic with solarnargine Binding of cholesterol Inhibition of GABA response in chemosensory cells Blocking of action potential Activation of Na+ channels

430,433 432 383 432 435 435 383 234 234,427

Quenching of singlet oxygen Binding to muscarinergic acetylcholine receptor Binding/inhibition of dopamine uptake carrier

361 312 12,436

Activation of Na+ channels, no repolarization Inhibition of RNA polymerases I1 and I11 (transcription) Modulation of acetylcholine receptor Binding to acetylcholine receptor Increase of Na' permeability Inhibition of Na+,K+-ATPase,glucose transport Inhibition of mitochondria1 electron transport Inhibition of Na+,K+-ATPase Inhibition of protein biosynthesis Alkylation of DNA Modulation of glycine neurochemical activity Inhibition of energy transfer in mitochondria Inhibition of energy transfer in chloroplasts Binding to auxin receptors in plants Inhibition of ATPase Inactivation of SH groups Inactivation of amino groups

259,427 376 230 437 234,388 439 440 408 404,444 343 364 445 106 106 106 446,447 446,448 (continued)

TABLE 1V (Continued) Alkaloid Gephyrotoxin Haningtonine Homoharringtonine Hemanthamine Hippeastrine Histrionicotoxin Irehdiamine Isoharringtonine Lycorine

pm

Maitoxin Malouetine Maytansine Maytansinine Methyl1ycaconitine C15-2,bmethylpiperidine Muscarine Narciclasine Nicotine

Ochratoxin Olivacine Palytoxin Pilocarpine Pretazettine Pseudo1ycorine

Effect Inhibition of acetylcholine receptor Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of DNA polymerase Inhibition of K+ channels Disturbance of membrane permeability Inhibition of protein biosynthesis Inhibition of DNA polymerase Inhibition of protein biosynthesis, binding to 60 S subunit Activation of CaZ+channels Disturbance of membrane permeability Binding to microtubules Inhibition of cell division Cholinergic agonist (insect nicotine receptor) Inhibition of mitochondria1 electron transport Inhibition of Na',K+-ATPase Binding to acetylcholine receptor Inhibition of protein biosynthesis Activation of acetylcholine receptor Inhibition of carotenoid biosynthesis Induction of vacuole formation in Puccinia Quenching of singlet oxygen Inhibition of glucose transport Intercalation with DNA Increase of Na+/K' permeability, hemolysis Binding to muscarinic acetylcholine receptor Inhibition of protein biosynthesis Inhibition of protein biosynthesis

Ref. 428 449 390 390 148 428 390 390 148 259,390 259 390 390 450 200 228 438 312 451 200,312 452 453 361 259 454 259 259 390 390

\o P

Psilocin/psiloc ybin F’umiliotoxin B Pumiliotoxin C Saxitoxin Solenopsine Streptonigrine Taxol Tetrodotoxin Trigonelline Tylocrebrine Tylocrepine Tylophorine Xestoaminol A, C Antibiotics Actinobolin Actinomycin Amphotericin B Bacitracin Bleomycin Calcirnycin Calichem ycin Cephalosporin Cephamycin Chloramphenicol Cycloheximide Cytochalasin B Daunom bicin Demecloc yclin

Interaction with serotonin receptor (hallucinogen) Inhibition of Ca2+channels Inhibition of acetylcholine receptor Inhibition of Na+ channels Inhibition of Nat,Kt-ATPase and mitochondria1 respiratory chain Inhibition of reverse transcriptase Promotion of polymerization of tubulin, polyploidization Inhibition of Na+ channels Promotion of cell arrest in G2of cell cycle in plants Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of protein biosynthesis Inhibition of reverse transcriptase

312 428 428 234,259 259 455 443 259,388 456,457 390 404 444 I I2

Inhibition of protein biosynthesis Intercalation in DNA, inhibition of RNA synthesis Interaction with membrane sterols, formation of membrane channels Inhibition of dolichol metabolism, geranyltransferase DNA binding and cleavage Inhibition of DNA polymerase, RNA polymerase, protein-glutamine y-glutamyltransferase CaZt ionophore in mitochondria DNA binding and cleavage Inhibition of transpeptidase Inhibition of transpeptidase Inhibition of translation Inhibition of translation Inhibition of glucose transport, blocking of contractile rnicrofilaments Inhibition of RNA polymerase, procollagen-proline,2-oxoglutarate 4-dioxygenase, intercalation with DNA Inhibition of translation

149 437 312 376 437 376 149 437 312 312 312 312 149,376,388 312,376 312 (continued)

TABLE IV (Continued) Alkaloid Doxorubicin Erythromycin Esparamycin Gentamycin Gramicidin Josamycin Kanamycin Lincomycin Mitomycin C Neomycin Novobiocin Nystatin A Oxytetracyclin Penicillins and p-lactam derivatives Polymyxins A-E Rifampicin Rifamycin Spectinomycin Spiramycin Streptomycin Tetracyclin Tobramycin Tyrothricin Vancomycin

Effect Inhibition of RNA polymerase, intercalation into DNA Inhibition of translation DNA binding and cleavage Inhibition of translation Formation of ion channels (Na', K+,H') in plasma membrane Inhibition of translation Inhibition of translation Inhibition of translation Alkylation of DNA, inhibition of replication phosphodiesterase Inhibition of I-phosphatidylinositol-4,5-biphosphate Inhibition of translation Inhibition of DNA topoisomerase Interaction with membrane sterols, formation of membrane channels Inhibition of translation Inhibition of transpeptidase (murein formation) Inhibition of protein kinase C, increase of membrane permeability Inhibition of DNA polymerase Inhibition of RNA and DNA polymerases Inhibition of translation Inhibition of translation Inhibition of translation Inhibition of translation Inhibition of translation Modulation of membrane permeability Inhibition of peptidoglycan biosynthesis

Ref. 312,376 312 437 312 312 312 312 312 312.437 312,376 376 312 312 312 312,376 376 376 312 312 312 312 312 312 312


51

a. Cellular Targets Nucleic Acids. DNA, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. It is not surprising that a number of secondary metabolites have been selected during evolution which interact with DNA or DNAprocessing enzymes. Some alkaloids bind to or intercalate with DNA/RNA (Table IV) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (Table V): 9-methoxyellipticine,dictamnine, ellipticine, harmane alkaloids, melinone F, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (PAS), cycasin, olivacine, etc. Many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of AT and GC base pairs. Other alkaloids act at the level of DNA and RNA polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. Whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (Table V). Besides basic data obtained in Salmonella or Drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. Anagyrine, anabasine, and coniine cause “crooked calf disease” if pregnant cows or sheep feed on these alkaloids during the first period of gestation (329,341,348,349,351,352).The offspring born show strong malformation of the legs. Some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by Veratrum species, cause the formation of a central large cyclopean eye (329-330, an observation that was probably made by the ancient Greeks and thus led to the mythical figure of the cyclops. It is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. In effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores. Another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (Tables IV and V). The alkaloid aristolochic acid, which is produced by plants of the genus Aristolochia, is carcinogenic. The mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine (344,345),because of its NO, group. Pyrrolizidine alkaloids and their N-oxides, which are abundantly produced by members of the Asteraceae and Boraginaceae but also occur in the families Apocynaceae, Celestraceae, Elaeocarpaceae, Euphorbiaceae, Fabaceae, Orchidaceae, Poaceae, Ranunculaceae, Rhizo-

TABLE V MUTAGENIC OR CARCINOGENIC ACTIVITYOF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Vinblastine/vincristine Vaocristine Quinoline alkaloids Dictamnine

Effect Fetal malformation in hamster Skeletal, ocular, and CNS malformations in man Mutagenic in yeast

Induction of revertants in Salmonella typhimurium (ST) Frameshift induction in E. coli Induction of revertants in ST Evolitrine Induction of revertants in ST Fagarine Induction of sister-chromatid exchanges Induction of revertants in ST Flindersiamine Induction of revertants in ST Kokusaginine Maculine Induction of revertants in ST Maculosidine Induction of revertants in ST Induction of revertants in ST Pteleine Induction of revertants in ST Skimmianine Alkaloids derived from phenylalanine/tyrosine Carcinogenic, mutagenic Aristolochic acid Mutagenic in ST Berberine Mutagenic Mutagenic in Lolium Colchicine Thebaine Teratogenic in hamster, congenital malformations Steroidal alkaloids 1 I-Deoxojervine (cyclopamine) Teratogenic, cyclopian malformation Jervine Teratogenic, cyclopian malformation Solanine Teratogenic in chick embryo, rumplessness

EDXI

50-100 pg/ml 5-20 &plate

-

5-20 pg/plate 5-20 &plate

5-20 5-20 5-20 5-20 5-20 5-20

pg/plate pg/plate pg/plate pglplate pglplate &plate

Ref. 324 325 284 326 327 326 326 328 326 326 326 326 326 326 344,345 346 297 347 260 329,330 329,330 261

Solasodine Veratrosine Pyrrolizidine alkaloids 7-Acet ylintermedine 7-Acety llycopsamine

Heliotrine Indicine Integenimine Intermedine Jacoline Lasiocarpine Lycopsamine Monocrotaline W 111

Retrorsine Senecionine Seneciphylline Senkirkine

Symphytine PAS general Quinolizidine alkaloids Anagyrine Cytisine

330.33I 329,330

Teratogenic, malformations in hamster embryos Teratogenic, cyclopian malformation Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Abdominal abnormalities in Drosophila Mutagenic in Drosophila Chromosome damage in mouse bone marrow cells Teratogenicity, mutagenicity Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in ST Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Mutagenic in Drosophila Chromosome breakagehearrangements in root tips Chromosome breakage in leukocytes Teratogenic, congenital malformations in calves (“crooked calf disease”) Teratogenic in chicks and rabbits

Minimal 0.01 mM Minimal 0.025 mM Minimal 0.025 mM 10 pM

Minimal 1 mM 18-38 mg/kg

Minimal 0.5 mM Minimal 0.1 mM

-

Minimal 1 mM 1 mM Minimal 0.0025 mM Minimal 0.05 mM Minimal 0.005 mM >I0 pM >10 pM

Minimal 0.005 mM Minimal 0.1 mM

333 333 332,333 334 333 336 336 337 333 333 338 333 332 333 333 333 335 335 333 333 339 340 329,341 341 (continued)

TABLE V Alkaloid Miscellaneous alkaloids Anabasine Arecaidine Caffeine Capsaicin Coniceine Coniine Cryptopleurine Cycasin DIBOA, DIMBOA Theobromine

(Continued)

Effect Teratogenic, crooked calf disease Chromatid exchanges in bone marrow cells Chromatid exchanges Mutagenic Teratogenic, congenital skeletal malformation in pigs Teratogenic, crooked calf disease Chromosome breaks in Drosophilu Mutagenic, carcinogenic Mutagenic in ST Genotoxicity Chromatid exchanges

ED%

Ref.

-

351 ,352 356 354 355 350 348,349 332 342,343 106 353 354

-

-


55

phoraceae, Santalaceae, Sapotaceae, and Scrophulariaceae (502)(-3% of higher plants produce these alkaloids), have mutagenic 2nd carcinogenic properties, provided the molecules have the 1,Zdehydro- l-hydroxymethyl-pyrrolizidine structure and are esterified (425,426).After oral intake, the N-oxides are reduced by bacteria in the gut. The lipophilic alkaloid base is resorbed and transported to the liver, where it is “detoxified” by microsomal enzymes. As a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. The alkaloid can then cross-link DNA and RNA and thus cause mutagenic or carcinogenic effects (especially in the liver) (502).Thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. The PA story is very intriguing, since it shows how ingenious Nature was in the “arms race.” The herbivores invented detoxifying enzymes, and Nature the compound which is activated by this process. A herbivore feeding on PA-containing plants will eventually die, usually without reproducing properly. Only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. The protection due to PA can easily be seen on meadows, where Senecio and other PA-containing plants are usually not taken by cows and sheep, at least as long other food is available. Protein biosynthesis. Protein biosynthesis is essential for all cells and thus another important target. Indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (Table IV), such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. For lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyltRNA with amino acids, as well as the elongation step. The inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected (503). Electron chains. The respiratory chain and ATP synthesis in mitochondria demand the controlled flux of electrons. This target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, CH,-(CH2),,-2,6-methyl-piperidines, capsaicin, the hydroxamic acid DIMBOA, and solenopsine. As mentioned before, however, only a few alkaloids have been evaluated in this context (Table V). Biomembranes and transport processes. A cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmenta-

56

MICHAEL WINK

tion that provides separated reaction chambers. Because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. The controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems. These complex systems are also targets of many natural products (Table IV). Disturbance of membrane stability is achieved by 9-methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. Steroidal alkaloids, such as solanine and tomatine, which are present in many members of the Solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky. Cells carefully control the homeostasis of their ion concentrations by the action of ion channels (Na+,K+,Ca2+channels) and through Na+,K+ATPase and Ca2+-ATPase.These channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. Inhibition of transport processes (ion channels, carriers) is achieved by (Table IV) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin. A special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (NA), serotonin, dopamine, glycine, and acetylcholine (ACH). We can distinguish two types. Type 1 is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ACH-receptor. In Type 2 the receptor is an integral protein. When a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent Gprotein molecule, which consists of three subunits. The a subunit then activates the enzyme adenylate cyclase, which in turn produces cAMP from ATP. The cAMP molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ACH receptor). A number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. Targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. Alkaloids relevant here include (Table IV) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, ga-


57

lanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine,anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. A number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly. Cytoskeleton. Many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. A number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (Table IV), namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. Colchicine, the major alkaloid of Colchicum autumnale (Liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. As a consequence, chromosomes are no longer separated, leading to polyploidy . Whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. Because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. The derivative colcemide is less toxic and can be employed in the treatment of certain cancers (312).Also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. For normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity. Colchicum plants are not attacked by herbivores to any substantial degree (185). Another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from Catharanthus roseus (Apocynaceae). These alkaloids also bind to tubulin (312).Both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias. From Taxus baccata (Taxaceae) the alkaloid taxol has been isolated. Taxol also affects the architecture of microtubules in inhibiting their disassembly (322). Nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin (312).In conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule. Enzyme inhibition. The inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see Table IV). Briefly, inhibition of CAMP metabolism (which is important for signal transduction I

58

MICHAEL WINK

and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by 1-ethyl-P-carboline, P-carboline-1-propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. Inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (Table IV). Castanospermine, swainsonine, and other polyhydroxyalkaloids are examples. b. Action at Organ Level. Whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too. Central nervous system and neuromuscular junction. A remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (Table IV). The cellular interactions have been discussed above. Disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects. A herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in Nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. Thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. Some individuals of Homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically. The activity of muscles is controlled by ACH and NA. It is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. In the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis. Alkaloids which activate neuromuscular action (so-called parasympathomimetics)include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. Inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) (312). Skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. The compounds are usually considered to be strong poisons, and it is obvious that

1. ALLELOCHEMICAL PROPERTIES OF

ALKALOIDS

59

they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare LD,, values in Table 11). Inhibition of digestive processes. Food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. The next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. The ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms (312). Another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. Relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (Table IV). Modulation of liver and kidney function. Nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. In the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. The liver is also the main site for detoxification of xenobiotics. Lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids (312).These conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination. Both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (Tables IV and V). The alkaloids are activated during the detoxification process, and this can lead to liver cancer. Also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins. Many alkaloids and other allelochemicals are known for their diuretic activity (312).For an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. Since Na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially. Disturbance of reproduction. Quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. Antihormonal effects could be achieved by mimicking the structure of sexual hormones. These effects are not known for alkaloids yet, but have been confirmed for other natural products. Estrogenic properties have been reported for coumarins, which di-

60

MICHAEL WINK

merize to dicoumarols, and isoflavones (4,17). Insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern Polypodium uulgare, or azadirachtin from the neem tree (4,17). Juvenile hormone is mimicked by a number of terpenes, present in some Coniferae. Spermatogenesis is reduced by gossypol from cottonseed oil (17). The next target is the gestation process itself. As outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo (Table V). The last step would be the premature abortion of the embryo. This dramatic activity has been reported for a number of allelochemicals, such as mono- and sesquiterpenes and alkaloids. Some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids (312). The antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. Nevertheless, they are defense strategies with long-term consequences. Blood and circulatory system. All animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. Inhibitors of the driving force for this process, the heart muscle, have already been discussed. However, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine (312). Some allelochemicals have hemolytic properties, such as saponins. If resorbed, these compounds complex membrane sterols and make the cells leaky. Steroidal alkaloids from Solanum or Veratrum species display this sort of activity as well as influencing ion channels (Table IV). Allergenic effects. A number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. Common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds (4,17,312). Activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. The function of alkaloids in this context is hardly known. This selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. However, most of the potential targets for the 10,000 alkaloids known at present remain to be established. If no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property.


61

Summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly. B. PLANT-MICROBE INTERACTIONS Dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay (7). How is this achieved? The aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products (4,7). This cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. In perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues. The only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. At the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas (4,17). In addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. Secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. These studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. It may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. In the pharmaceutical context we search for very active compounds which can be employed at low concentrations. Therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. These precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids. Secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono- and disesquiterpenes, and last but not least, alkaloids (4,17,42,149,322). In Table VI 183 alkaloids are tabulated for which antibacterial activities have been detected. The alkaloids usually affect more gram-positive than gram-negative bacteria. Especially well represented are alkaloids which

TABLE VI WITH ANTIBACTERIAL PROPERTIES ALKALOIDS Active against Alkaloid Alkaloids derived from tryptophan Athisine Ajmalicine Apparicine Aspidospermine Bisnordihydrotoxiferine

3

Bisnordihydrotoxiferine N-oxide Borreverine Brevicolline 5-Bromo-N,N-dimethylaminoethyltryptamine Brucine Bufotenine Canthin-Gone Caracurine V Caracurine V di-N-oxide 1-Carbomethoxy-P-carboline Catharanthine Cinchonine Cinchophylline Conoduramine Conodurine Cryptolepine 1GDecarbomethoxytetrahydrosecamine 18,19-Dehydro-ochrolifuanineF

Gram (+)

Gram (-)

Test

+

+ + + + + +

AL AD AD AL AD AL AD AL

+ + + + + + + +

+ +

-

+ + + + + + + + + + + + + +

+ + + + + + -

+

AD AD LD LD AD AD AD AD SP AD AD AD AD AD

Concentration tested (pg/m) I 15 12 1

MIC (mg/ml)

ED, (mdml)

lo00

lo00 270-3000 1-100

2 2-400

5 1 12-100 2 10- 1400 2 50 1 16-32 15-400 4-400

Ref. 50 51 52 50 53 54 53 55 57 72 53 56 113 50,58 53 53 41 51 56 69 59-60 59.60 61 42,43 69

Dehydropteleatinium Dictamnine Dihydrocinchonine 18,19-DihydrocinchophyIline Dihydrocorynantheol 4,5-Dimethoxycanthin-6-one 10,IO'-Dimethoxy-Nmethyltetrahydrousambarensine Diploceline Fagarine Glycozolidol Gramine Harmaline

W QI

+ + + +

+ + + + + + + +

Harmalol Harman Harmine

+ +

Harmol 3-Hydrox yconoduramine 3-Hydroxyconodurine 3-H ydroxyconopharyngine 3-H ydroxy isovoacangine 3'-Hydroxy-p-demethylervahanine B 3'Hydroxy-N"-demethyItabernamine 19-Hydroxy-18,19-dihydrocinchophylline 9-Hydroxyellipticine 3-Hydroxy-(19R)-heyneanine 5-Hydroxy-4-methoxycanthin-6-one

+ + + + + + + + + + +

+

AL

50-100 10

BG

-

94 95

AL AD BG

50 69 42,62,63 41 69

AD

+ + + + + + + + + + + + + +

BG AD LD PD SP SP PD SP

500-2000 20 200

4

10 (light)

I 4 10 (light)

< 100 SP AD AD AL AL BG BG AD AD BG

10 (light)

8-170 14-750 60-140 50-500

1-250

64 95 65 113 67 66 56 67 66 68 66 68 45 45 47 45 44 44 69 48,49 44 41 ~

(continued)

TABLE VI (Continued) Active against Alkaloid

10'-Hydroxy-10-methoxy-Nmethyltetrahydrousambarensine 3'-Hydroxytabernamine 16Hydroxytetrahydrosecamine 10-Hydroxyusambarensine 3-Hydrox yvoacamine

2

Ibogaine Ibogamine Iboxygaine Isoraunescine Isovoacangine Melicopicine 6-Methox ytecleanthine

11-Methoxytubotaiwine Mimosamycin Norharmane Ochrolifuanine A Ochrolifuanine E Ochrolifuanine F Perivine Pteleatinium Ptelefolonium Renierol Reserpine Stemmadine Strychnine

Gram ( + )

+ + + + + + + + + + + + + +

+ + + + + + + +

-

+

Gram (-)

+ +

-

+ + + + -

+ +

Test

Concentration tested (pg/m)

MIC (mg/ml)

ED, (mg/ml)

Ref.

AD

69

BG BG AD BG AD AL AL AL AL AD AD AD

44 43,43 69 45 46 50 50 50 50 97 97 42.43 93 66 43,69 69 69 51 94,96 42 93 51 70 53 56

50 1 1 1

1

lo00 lo00 lo00 lo00 >200 >200 100

SP BG AF AF AD AL

10 (light) 32 32 15 100-lo00 100

AD

SP AD SP

37 1-7 5

1

c

Tabernaemontanine Tabernanthine Tchibangensine Tecleanthine Tetrahydroalstonine Tetrahydrosecamine Tetrahydrousambarensine Usambarensine Vindoline Vindolinine Voacamine Vobparicine Vobparicine N-oxide Woodinine Yuehchukene Alkaloids derived from phenylalanine/tyrosine Actinodaphnine Anhydroushinsunine Anolobine Anonaine Berbamine Berberine Berberrubine Bulbocapnine Cassameridine Cepharanthine Chelerythrine

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

+

+ -

+ + + + +

AD AL AD AD AD AD AD AD AD AD AD AD BG AD

38 1

51 50

lo00 64 >200

69 97 51 62.63 69 69 51 51 60 45 45 72 71

54 110 32 38 70 20-400 50- 100

20-25

+ + + + + + -

+ +

AL AL AL AL AL AL AL SP AL AL AL AL

50-300 50 6-200 3-100 50 125-1000 1000 3-100 1

100 1000 25-50 8-1000 6-100

74 74 80,81 80,81 74 73 50 89 56

90 50

82 73 50,84,85 (continued)

TABLE VI (Continued) Active against Alkaloid Chelidonine

Chelidonine N-oxide Columbamine Corytuberine I-Curine Dehatrine Dehydroglaucine Dihydroberberine Fagaronine Funiferine Glaucine Hernandezine Isoboldine Isotetrandine Isotrilobine Liriodenine Lysicamine Magnoflorine N-Desmethylthalidezine N-Desmethylthalistyline N-Methylactinodaphnine Nornantenine Nuciferine

Gram (+)

Gram (-1

Test AL AL AL AL AL AL AL AL AL AL AL

Concentration tested (pglm)

MIC (mg/ml) 1000 1000- 10,000 20-50 30-500 1000-10,000 100 1000 300 25 1000

SP AL AL AL AL AL AL

SP AL AL AL AL AL AL AL

100 300 25-100

100-200 8-500 1-100 0.4-3 12-26 50-1000 100-1000 50-300 3-100

loo0

ED, (mglml)

Ref. 50 86 87 42 86 79 80,81 50 74 83 50 88 50 74 75 80,81 76 73 80,81 83 82 78,79 75 75 74 80,81 50

0-Methyldauricine 0-Methylthalibrine 0-Methylthalmethine Obamegine Oxonantenine Oxyacanthine Palmatine Papaverine Pennsylvanine Protopine Protothalipine Sanguinarine

3

Tetrandrine Thalibrine Thalicarpine Thalicerbine Thalidasine Thalidezine Thaliglucinone Thalistine Thalistyline Thalmelatine Thalmirabine Thalphenine Thalrugosaminine Thalrugosidine Thalrugosine Tubocurarine

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

-

+ + + -

+ + -

+ + + +

-

-

+ +

250- lo00

AL AL

AL AL

AL

SP AL AL AL AL AL

AL SP

100 100

50-200 6-25 62-100 lo00

I lo00 100

300 lo00 13-100 1-5

84 0.01

15-1000

AL AL AL AL AL AL AL AL AL AL AL AL AL SP

73 77 75 76 82 73 50 56 75 87 74 79

lo00 100-1000 250- I 000

25-200 100

25-200 100

50 100 100 1000 50-100

100-200 100-200 1

87 56 73 75 42,78 73 76 75 79 77 75 42,78 77 78,79 78,79 76 76 56 (continued)

TABLE VI (Continued) Active against Alkaloid Xylopine Steroidal alkaloids Conessine Samandarone Samandarine Solacasine Solanidine Solanocapsine Quinolizidine alkaloids Angustifoline 13-Hydroxylupanine Lupanine Sparteine 13-Tigloyloxylupanine Pyrrolizidine alkaloids Lasicarpine Miscellaneous alkaloids Antofine

Gram ( + )

+ + + + + + + + + + + + + + +

Gram (-)

Test

Concentration tested (pg/m)

MIC (mg/ml)

AL

50-100

AL LD LD AL AL AL AL

100-1Ooo

ED, (mg/ml)

74 50 113 113 50 50 91 91

3-13 loo0 3 100 50 mM 50 mM 50 mM

SP AD

I00 40 1-40 2-22 15

>I00

125 125 125 129,130 125 113,529 113,529 113,529 121 124 126,127 126 126,127 124 126,127 42 128 126,127 121 128 126,127 121 121 126,127 121 124 126,127 121,126,127 126,127 126 124 (continued)

TABLE VII (Continued) Alkaloid Tomatine

rn 4

Veratramine Veratridine Veratrobasine Verazine Quinolizidine alkaloids Lupanine Sparteine 13-Tigloyloxylupanine Miscellaneous alkaloids Antofine Benzoxazolinone (BOA) Cryptopleurine Dictamnine 6,6’-Dihydroxythiobinupharidine DIMBOAlMBOA 3,4-Dimethoxy-(piperid2-yl)-acetophenone

Active against

Test

Fungi Fungi Yeast, fungi Fungi Yeast, fungi Yeast, fungi

CT TLC

Etysiphe graminis Etysiphe graminis Fungi Erysiphe graminis

AL AL AL AL

Fungi Fungi Candida, fungi Fungi Fungi Phytopathogenic fungi Candida

CT AL

Concentration tested (mdml)

MIC (pg/ml)

EDXI (mg/ml)

2-40 5 72-200

126,127 42,126 129,130 125 129,130 124

1

72-200 3-12 2 mM loo

37

50 TLC AL

3-6 0.1

94,131 95 110 95 70 100 97 104 107 137

* CT, Channel test according to Wolters ( I 16); other abbreviations are as in Table VI. If a range is given, the first value gives a 10% inhibition, the second value a 100% inhibition.

78

MICHAEL WINK

specialized on a particular host plant. However, alkaloid production does not necessarily have to be involved with antimicrobial defense. For example, Phytophthora or Fusarium will attack alkaloid-rich plants of Nicotiana, Solanum esculentum, and S . tuberosum. Cladosporium and Fusarium can develop in nutrient-containing media enriched with alkaloids, and Aspergillus niger can utilize alkaloids as a nitrogen source (506). In addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. In these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. We suggest that these specialists have adapted to the chemistry of their host plants. Mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). These mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. Some associations between plants and fungi are symbiotic in nature, such as Rhizobia in root nodules of legumes or microrhizal fungi in many species. In lupines, nitrogen-fixing Rhizobia are present both in alkaloid-rich and alkaloid-free plants. They must therefore be able to tolerate the alkaloids, which are also present in the root. Alkaloid production in lupines is more or less unaffected whether or not the plants harbor Rhizobia (185,506). An ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. Graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi (4). A similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity. From the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (Tables I1 and 111). Since many alkaloids are antibacterial and antifungal (Tables VI and VII) and are present in plants at relatively high concentrations (Section IILA), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. It should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes (2-4,7). A cooperative, or even synergistic, process could thus be operating.

1. ALLELOCHEMICAL PROPERTIES

OF ALKALOIDS

79

C. ANTIVIRALPROPERTIES Plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (Heteroptera). Resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place. The assessment of antiviral activity is relatively difficult. As a result, only a few investigators have studied the influence of alkaloids on virus multiplication. Nevertheless, at least 45 alkaloids have been reported with antiviral properties (Table VIII). Only sparteine (527) and cinchonidine (142) have been tested for antiviral activities against a plant virus, the potato X virus. All other evidence for antiviral activities (Table VIII) of alkaloids comes from experiments with animal viruses. Because viral life strategies are related in plants and animals, we suggest that a wider number of plant viruses may be controlled by alkaloids in Nature than the limited data imply. Viral multiplication can be controlled at the level of replication, transcription, protein biosynthesis, and posttranslational protein modification. The number of molecular targets is thus quite restricted for antiviral activities (compare Tables IV and VIII). The processing of DNA and RNA is extremely important for viruses, and it is not surprising that this area (intercalation in DNA, binding to DNA, inhibition of RNA and DNA polymerases) is probably one of the potential targets of alkaloids, for example, camptothecine (365,366),quinine, p-carboline alkaloids (1381, and acridone alkaloids (145). Other alkaloids could inhibit protein biosynthesis or posttranslational protein modifications. Examples include polyhydroxy alkaloids (150,212,410-414), cryptopleurine (404,444), haemanthamine (390),hippeastrine (148), narciclasine (451), pretazettine (390), sparteine and other QAs, and pseudolycorine (390). Because retroviruses rely on reverse transcriptase, inhibition of this enzyme by alkaloids would have a dramatic effect. However, plant viruses are not retroviruses, and the significance of the anti-reverse transcriptase effects of the alkaloids listed in Table VIII are difficult to interpret at present. Polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum- and Golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins. Because alkaloids often deter the feeding of insects, such as aphids and bugs (Table I), viral infection rates may be reduced in alkaloid-rich plants. Such a correlation exists for alkaloid-rich lupines (so-called bitter

TABLE VIII ANTIVIRAL ACTIVITY OF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Apparicine Camptothecine Cinchonidine

Dimethoxy-1-vinyl-P-carboline Eudistomins C, E, K, L (tunicates) Harman Harmine 7-Methoxy-I-methyl-P-carboline Norharman Alkaloids derived from phenylalanineltyrosine Fagaronine Acridone alkaloids Acronycine Atalaphillidine Atalaphillinine Citpressine I Citracridone I Citrusinine I Dercitine (sponge) Dimethox yacronycine Glycocitrine I GIyfoline Grandisine

ED, (dnl)

Activity

Ref.

Anti-polio I11 activity Inhibition of herpes and other virus Inhibition of potato X virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition of murine cytomegalovirus, Sindbis virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus

-

-

141 140 142 138 109 138 139 138 138

Inhibition of reverse transcriptase of oncorna virus

-

143

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition

3.3 0.7 0.8 0.6 1.3 0.7 1-5

145 145 145 145 145 145 144 145 145 145 145

of of of of of of of of of of of

herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes

simplex virus simplex virus simplex virus simplex virus simplex virus simplex virus simplex virus, murine corona virus simplex virus simplex virus simplex virus simplex virus

-

6.5 5

>20 10

5-Hydrox y-N-methylseverifoline 5-H ydroxynoracronycine 5-Methox yacronycine N-Methylatalaphilline Miscellaneous alkaloids Abikoviromycin Ageliferin Crinamine Cryptopleurine Sceptrin Didemnin Haemant hamine Hippeas trine 6-H ydroxycrinamine Ly corine

May tansine Narciclasine Oxysceptrine Precriwelline Pretazettine Pseudo1ycorine Sparteine Polyhydroxy alkaloids Castanospermine Deox ynorjirimycin Dihydroxymethyl-dihydrox ypyrrolidine

" MAD, Minimal active dose.

Inhibition of Inhibition of Inhibition of Inhibition of

herpes simplex virus herpes simplex virus herpes simplex virus herpes simplex virus

2.0 5 5.5 8.4

145 145

Antiviral activities Inhibition of herpes simplex virus Inhibition of Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition of murine sarcoma virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition to Rauscher virus NIH/3T3 cells Inhibition of herpes simplex virus Inhibition of potato x virus

-

149 109 147 141 109 109 147 148 147 147 148 146 147 109 147 147 148 147 148 150

Inhibition of cytomegalovirus, retroviruses Inhibition of cytomegalovirus, retroviruses Inhibition of cytomegalovirus, retroviruses

0.8 mM 1.0 mM 1.8 mM

M A P 0.2 pg/ml

-

MAD 0.2 pg/ml

-

MAD 0.2 pg/ml MAD 0.2 pg/ml -

MAD 0.005 pg/ml

-

MAD 0.05 pg/ml

-

MAD 1.0 pg/ml

-

i45 145

150 150 150

82

MICHAEL W I N K

lupines) and low-alkaloid varieties (the so-called sweet lupines) (see Table XII).

D. ALLELOPATHIC PROPERTIES Plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. This phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense (4,17,498-500). A number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated. It was observed by Molisch in 1937 (497) that plants can also influence each other by their constituent natural products, and he coined the term “allelopathy” for this process. Secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain (4,17). Both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. Allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments (4,17,19,497-500),but how it operates in ecosystems is still often a matter of controversy. It is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. Thus allelochemicals might never reach concentrations high enough to be allelopathic. Allelopathic natural products have been recorded in all classes of secondary metabolites. Few research groups have studied the effect of alkaloids in this context, but at least 50 alkaloids have been reported with allelopathic properties (Table IX). As can be seen from Table IX, allelopathic activities can be found within nearly all structural types of alkaloids. At higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. More sensitive, however, is the growth of the radicle and hypocotyl. They respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. In species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in Colchicum autumnale (503,506). It is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms.

TABLE IX ALLELOPATHIC ACTIVITY OF ALKALOIDS Alkaloid Alkaloids derived from tryptophan Quinine Cinchonidine Cinchonine Ergometrine Ergotamine Grarnine

Harmaline Hordenine 5-H ydroxytryptophan

Physostigmine Quinidine

Strychnine

Activity Toxic to Cinchona cells Toxic for Lemna Toxic to Cinchona cells Toxic for Lemna Toxic to Cinchona cells Reduction of radicle length in Lepidium, Lacruca Reduction of radicle length in Lepidium Reduction of radicle length in Lepidium Reduction of radicle length in barley Growth inhibition of Stellaria, Capsella. Nicotiana Reduction of radicle length in Lepidium, Lactuca Reduction of radicle length in Lepidium Toxic for Lemna Reduction of radicle length in barley Growth inhibition Toxic for Lemna Inhibition of germination Toxic to Cinchona cells Reduction of radicle length in Lepidium, Lactuca Toxic for Lemna Reduction of radicle length in Lepidium Toxic for Lemna Toxic for Lemna

Yohimbine Alkaloids derived from phenylalanine/tyrosine Reduction of radicle length in Lepidium. Lactiica Berberine Growth inhibition in plant cell cultures

Ref. 0.04% 0.04% -

0.4%

244 56 244 56 244 56 56 56 239 240 56 56 56 239 238 56 24 I 244 56 56 56 56 56

0.01% -

56 243

0.01% 0.1% 0.1%

-

0. I% 0.01% 0.04%

0.4% 0.1-0.0 1% 0.4% 0.1% 0.4%

(continued)

TABLE IX (Continued) Alkaloid Boldine Chelidonine Colchicine Emetine Ephedrine Morphine Narcotine Papaverine Salsoline Sanguinarine

g

Tropane alkaloids Cocaine H yoscyamine

Scopolamine

Quinolizidine alkaloids Cytisine Lupanine Sparteine

13-Tigloyloxylupanine

Activity

ED54

Toxic for Lemna Reduction of radicle length in Lepidium Reduction of radicle length in Lepidium Toxic for Lemna Reduction of radicle length in Lepidium Reduction of root growth, induction of polyploidy in Allium Inhibition of germination Reduction of root growth, induction of polyploidy in Allium Reduction of radicle length in Lepidium Reduction of radicle length in Lepidium, Lactuca Toxic for Lemna

0.04% 0.1% 0.01% 0.4% 0.1%

Inhibition of germination Inhibition of germination, radicle growth in Linum Toxic for Lemna Inhibition of germination Inhibition of germination, radicle growth in Linum, wheat Reduction of radicle growth in Lactuca Inhibition of germination

0.4% -

Reduction of radicle length in Lepidium Inhibition of seed germination in Lactuca Inhibition of seed germination in Lacruca Reduction of radicle length in Lepidium, Lactuca Inhibition of seed germination in Lactuca Inhibition of radicle growth in Raphanus Inhibition of radicle growth in Sinapis Inhibition of seed germination in Lactuca

0. I% 6 mM m

172

ARNOLD BROSSI

HO

()$OH

H

CH3

CH3

SAL

0

H

O

~

N

H

+

H

C"3

E

CH3

F

\ o m \N

C

H O & N H +

CH3

A

H

O

~

CH3

B FIG.42. Electrochemical oxidation of racemic salsolinol (SAL).

CH3

D

VII. Conclusions Endogenous mammalian alkaloids occur in tissues and fluids at very low levels, and none has ever been isolated in amounts sufficient to allow determination of optical properties by direct measurements. Biological testing in uitro and in uiuo was performed almost entirely with racemic mixtures composed of optical isomers with different biological profiles. A recent report that optically active tetrahydroharmine racemized in the presence of acid suggests that TBCs may be subjected to racemization during workup and afford partially racemized material. For this reason it is difficult to judge whether the biological data reported for mammalian alkaloids are real or unreliable owing to optical inhomogenity. This field of research, although exciting and important in connection with problems associated with alcohol addiction, drug abuse, and disorders originating from interference with dopamine and serotonin formation and metabolism, in the opinion of the author, will only mature and become meaningful when appropriate standards and controls are implemented. A close collaboration of biochemists and clinicians with organic chemists is highly recommended so that research can result in medically meaningful advances.

Acknowledgments

The interest in the National Institutes of Health mammalian alkaloid program by Dr. Bernhard Witkop, NIH Institute Scholar, and his help in finalizing this review are gratefully acknowledged.

N

2.

MAMMALIAN ALKALOIDS I1

173

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t

AMPHIBIAN ALKALOIDS JOHN W. DALY,H . MARTINGARRAFFO, A N D THOMAS F. SPANDE Laboratory of Bioorganic Chemistry National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda. Maryland 20892

.....................................

187

111. Bicyclic Alkaloids ............................................................................. A . Histrionicotoxins (Azaspiro[5.5]undecanols)...................................... B. Decahydroquinolines ...............................

199 200

B. Samandarines .........................................

D. Pyrrolizidines .........

....................................

225

F. Quinolizidines ..................................................................... IV. Tricyclic Alkaloids ....... A. Gephyrotoxins ............................................................................. B. Coccinellines .................................................................... C. Cyclopenta[h idines ..................... D. Pyrrolizidine .................................................................... V. Monocyclic Alkaloids ........................................................................

249 251

....................................

255

.......................................

251 261

V1. Pyridine Alkaloids A. Epibatidine ............................................ V11. lndole Alkaloids ..... A. Pseudophrynamines B. lndole Amines C. Dehydrobufote

....................................

238 242 245

...........................

VIII. Imidazole Alkaloids .................................... 263 IX. Morphine .................................................... X. Guanidinium Alkaloids ....................................................................... 264 A . Tetrodotoxin .......... .................. 264

C. Zetekitoxin .............. XI. Other Alkaloids ........................................... I85

.....................................

268

THE ALKALOIDS. VOL.. 43

186

JOHN W . D A L Y E T A L .

XII. Summary .........................................................................................

275 Appendix ...... 277 References ....................................................................................... 281

I. Introduction

Amphibians have developed a wide range of biologically active compounds that are present in skin, often in relatively large amounts. It would appear that many such compounds serve as chemical defense, being released onto the skin surface from cutaneous granular (poison) glands. These compounds include biogenic amines, peptides, proteins, the steroidal bufadienolides and cardenolides, and more than two dozen classes of alkaloids. A general taxonomic survey of the occurrence of such noxious/ toxic substances in skin of amphibians was presented in 1987 (I). Many of these compounds, because of noxious or toxic effects on nerves and muscles of buccal tissue, clearly could serve the host amphibian in defense against predators. Certainly among the amphibian alkaloids, very potent neurotoxins, such as batrachotoxins, samandarines, and tetrodotoxin, are admirably suited as chemical defenses. However, among the peptides there are certain examples, such as the magainins, that in view of potent antimicrobial activity probably serve as a defense against infections from protozoans, fungi, and bacteria (2). Consonant with the hypothesis (see discussion in Ref. 1 ) that the unusual secondary metabolites present in amphibian skin serve in chemical defense against predators and/or microorganisms, most such compounds, including the many classes of alkaloids, exhibit marked biological activity. The uniqueness of the structures of the different alkaloids, which often occur only in a single genus, has focused attention on their taxonomic significance and biosynthetic source. Such amphibian alkaloids also have afforded a challenge for chemical synthesis. A number of reviews in the last decade (3-5)have focused on synthesis of amphibian alkaloids. The present chapter will not treat synthesis but will document only the structural diversity, spectral and chemical properties, biological activity, and distribution in Nature of the nearly 300 known amphibian alkaloids.

3. AMPHIBIAN ALKALOIDS

187

11. Steroidal Alkaloids

Two classes of steroidal alkaloids have been discovered in amphibians, the batrachotoxins and the samandarines. Both discoveries had as their starting point the folk knowledge that a brightly colored amphibian was poisonous. In the case of batrachotoxins, it was the knowledge of Indians that skin secretions of certain brightly colored frogs native to rain forests west of the Andes in Colombia were sufficiently toxic to be used in poison blow darts (see Refs. 5 and 6 for reviews of early literature). Studies initiated at NIH in 1962 (6) led ultimately to the isolation and structure elucidation of the unique steroidal alkaloid batrachotoxin and several congeners. The existence of amphibian alkaloids, indeed of alkaloids from an animal rather than a plant source, had been established in 1866 (71) during investigations of the European fire salamander, a brilliant black and yellow amphibian known since ancient times to be poisonous. The characterization of the steroidal alkaloid samandarine and its congeners from this salamander was initiated in the 1930s, leading, over the following 30 years, to the structural elucidation of all the major alkaloids of the samandarine class (8-10). The batrachotoxins and samandarines were major challenges from the standpoint of structure elucidation, and X-ray crystallographic analysis played a major role.

A. BATRACHOTOXINS I . Structures

The isolation and characterization of batrachotoxin and its congeners from extracts of the skin of the poison-dart frog Phyllobates aurotaenia (6,11-13), and later from the poison-dart frog Phyllobates terribilis ( 1 4 , have been reviewed in detail (5). The key events were the preparation of a crystalline 4-bromobenzoate of a less toxic but stable congener, batrachotoxinin A, and X-ray analysis of this derivative (12), followed by a reevaluation of the spectral properties of the much more toxic batrachotoxin and its major congener, homobatrachotoxin (13). It should be noted that, because of early incorrect interpretations of mass spectra, homobatrachotoxin was initially referred to as “isobatrachotoxin” (12). X-Ray analysis of the 4-bromobenzoate demonstrated that the structure of batrachotoxinin A is 3a,9a-epoxy-l4P, 18-(2’-oxyethyl-N-methylamino)-5Ppregna-7, 16-diene-3/?,1la,20a-triol, as shown in I.

188

JOHN W . DALY E T A L .

OH

HO

Batrachotoxinin A (1)

The absolute configuration at C-14 differs from cholesterol, and instead is reminiscent of the bufadienolides and cardenolides. The conformation is markedly constrained by the 3,9-oxygen bridge and the homomorpholine bridge at the C,D ringjuncture. The reevaluation of the spectral properties and comparison to model ethyl pyrrole 2- and 3-carboxylates led to the conclusion that batrachotoxin was the 20a-2,4-dimethylpyrrole-3carboxylate, and that homobatrachotoxin was the 20a-2-ethyl-4-methyIpyrrole-3-carboxylate. The structure of batrachotoxin was confirmed by acylation of the 20a-hydroxyl group of batrachotoxinin A with a mixed anhydride from 2,4-dimethylpyrrole-3-carboxylicacid (13). The structures of batrachotoxinin A, batrachotoxin, homobatrachotoxin, and the 4phydroxy congeners of batrachotoxin and homobatrachotoxin are shown in Fig. 1. The structure of pseudobatrachotoxin, a labile congener that yields batrachotoxinin A during storage, remains unknown. Chemical properties of the batrachotoxins were assessed initially on a microscale (see review in Ref. 5 ) . The Ehrlich reaction proved to be a sensitive indicator of the presence of a pyrrole moiety. Physical and spectral properties of batrachotoxins are presented in Table I. Mass spectra have been presented and interpreted (3,13,14). The parent ion of batrachotoxin is virtually nondetectable by direct probe methods, and instead an apparent molecular ion of m l z 399 is seen, probably because of pyrolytic elimination of the pyrrole carboxylate moiety. Batrachotoxin alkaloids do not chromatograph on capillary gas chromatographic columns, but a pyrolysis product has been detected at 280°C on the temperature-programmed, packed OV-1 columns used for analysis of other dendrobatid alkaloids (see Appendix). The pyrrole carboxylate moiety is responsible for major ions of C,H9N02+ ( m l z 139), C,H9N+

3. AMPHIBIAN

I89

ALKALOIDS

1

R=

H

H

OH H

FIG.1 . Structures of batrachotoxinin A (A, R

H), batrachotoxin (A, R = 2), homobatrachotoxin (A, R = 3), 4P-hydroxybatrachotoxin (B, R = 2). and 4P-hydroxyhomobatrachotoxin (B, R = 3). =

(m/z95), and C,H,N+ (mlz94) in batrachotoxin and C,H,,NO,+ (mlz 153), C7H,N02+ (mlz 138), C 7 H I , N +(mlz 109), and C6H8N+(mlz 94) in homo-

batrachotoxin. The homomorpholine ring yields a major fragment ion of C,H,,NO+ (mlz 88). Proton magnetic resonance spectra have been presented and discussed (3,5,13-15). Carbon- 13 magnetic resonance assignments have also been reported (14).

2 . Biological Activity Batrachotoxin, not unexpectedly in view of its use as a dart poison by South American Indians, is an extremely toxic substance. The LD,, on subcutaneous injection in mice is about 40 ng. Homobatrachotoxin is only slightly less toxic, while batrachotoxinin A is 500-fold less toxic. The nature of the ester function at the 20a position is of critical importance to toxicity. Thus, the 20a-benzoate of batrachotoxinin A is fully as toxic as batrachotoxin, whereas the 20a-4-bromobenzoate has very low toxicity. For a summary of the toxicities of natural and synthetic batrachotoxins, see Ref. 5 . The pharmacology of batrachotoxins that underlies their high toxicity is the result of a specific high-affinity interaction of batrachotoxin with a

190


TABLE I PHYSICALA N D SPECTRAL PROPERTIES OF BATRACHOTOXINS ( 5 , lI, 13-16) Batrachotoxin, C31H42N206 Mass spectrum: mlz 538 (el),399(3), 312(13), 294(10), 286(10), 184(30), 139(65), 138(24), 122(20), 121(13), 120(10), 109(15), 95(100), 94(100), 88(34), 71(26) Ultraviolet: A, 234 nm, E 9800: 262 nm, E 5000 Infrared: 1690 cm - I Optical rotation: [a]:& - 5 to - lo", [a]&-260" (0.23, CH,OH) Rfvalue: 0.45' Homobatrachotoxin, C32HuN206 Mass spectrum: rnlz 552(41), 399(6), 312(25), 294(20), 286(22), 184(60), 153(90), 139(22), 138(100), 122(12), 121(15). 120(26), 109(60), 95(23), 94(94), 88(72), 72(28) Ultraviolet: A,,233 nm. E 8900, 264 nm, E 5000 Infrared: 1690 cm - I Rfvalue: 0.50 4P-Hydroxybatrachotoxin, C3,H42N207 Mass spectrum: rnlz 554 (el),415(6), 386(5), 328(30), 310(18), 184(38), 139(68), 83100) Rfvalue: 0.10

4~-Hydroxyhomobatrachotoxin,CI2HMN2O7 Massspectrum: mlz568(~1),415(5), 386(6). 328(7),310(17), 184(14),153(60),139(100),8466) Rr value: 0.10 Batrachotoxinin A, C24H35N05 Mass spectrum: mlz 417(2), 399(11), 330(100), 312(30), 202(15), 184(11), 158(14), 88(60) mp: 160-162°C (synthetic) Ultraviolet: end absorption Optical rotation (synthetic): [a]! - 42" (0.45, CHIOH) Rfvalue: 0.28 pK,: 8.2 Pseudobatrachotoxin' Mass spectrum: mlz 399(6), 312(17), 294(15), 286(6), 202(3), 184(48),88(100),71(45); ions at mlz 342 (C22H32N02. 6) and 166 (CIOHl6NO, 12) were detected, but are probably due to an impurity Ultraviolet: end absorption Rfvalue: 0.25 a Ultraviolet spectra are shown in Ref. 13. 'Thin-layer chromatography: silica gel, HCCI,-MeOH (9: I , vlv). Pseudobatrachotoxin converts to batrachotoxinin A at room temperature.


191

site on voltage-dependent sodium channels of nerve and muscle. Interaction of batrachotoxin with this site stabilizes the sodium channel in an open formation, leading to a massive influx of sodium ions and depolarization of nerve and muscle. Tetrodotoxin, through a specific blockade of sodium channels, can prevent batrachotoxin-elicited depolarization. The activation of sodium channels by batrachotoxin is time- and stimulus-dependent, suggesting that batrachotoxin acts preferentially on an open channel. Certain other alkaloids, namely, veratridine and aconitine, and the diterpene grayanotoxin, appear to interact at the same site as batrachotoxin, but they are much less potent and efficacious in maintaining the channel in an open, conducting form. The action of batrachotoxin on sodium channels can be enhanced allosterically by certain polypeptide neurotoxins, namely, a-scorpion toxin and anemone toxins, and by the terpenoid brevetoxins from marine organisms. Conversely, many local anesthetics appear to reduce allosterically the action of batrachotoxin. The development of a batrachotoxinin A 20a-[3H]benzoate as a radioligand for batrachotoxin-binding sites on sodium channels has proved invaluable in studying the allosteric regulation of such interactions. Batrachotoxin at present remains an important, indeed often essential, tool for mechanistic studies of the function of voltage-dependent sodium channels and for the investigation of the role of depolarization and/or influx of sodium ions on physiological functions. Batrachotoxin has been particularly useful in the study of the function of sodium channels, purified and reconstituted into artificial lipid bilayers. A summary and overview of the extensive studies with batrachotoxin appeared in 1986 (5). Since that time more than 100 articles dealing with the activity of batrachotoxin and/ or the radioligand batrachotoxinin A 20a-[3H]benzoate have appeared, and it is beyond the scope of the present review to summarize this extensive recent literature. A few selected developments are as follows: allosteric enhancement of the action of batrachotoxins by pyrethroid insecticides (17,18) and inhibition by polyunsaturated alkanoic N-alkylamide insecticides (e.g., pelliterine) (19);allosteric enhancement of binding of batrachotoxinin A 20a-[3H]benzoateby the polypeptide striatoxin isolated from a marine snail (20); stimulation of phosphoinositide breakdown by batrachotoxin and dependence on influx of sodium ions (21); structure-activity relationships for derivatives of 7,8-dihydrobatrachotoxininA (22); an apparent interaction of muscarinic receptors and sodium channels that can affect the binding and action of batrachotoxin (23);development of irreversible blockers of binding batrachotoxinin A 20a-[3H]benzoate(24,25); demonstration of cyclic AMP-dependent regulation of binding sites for batrachotoxinin A 20a-[3H]benzoate(26).

192


3. Occurrence

The batrachotoxins represent a unique class of steroidal alkaloids whose structures contain several unprecedented elements, in particular the homomorpholine ring sharing the steroidal C,D ring juncture, the 2,4-dialkyl pyrrole-3-carboxylate moieties, and the 3,9a-hemiketal oxygen bridge. There is, to our knowledge, no other natural compound closely related in structure to the batrachotoxins, and hence the biosynthetic origin is of some interest. A preliminary study revealed no detectable incorporation of radiolabeled acetate, mevalonate, cholesterol, or serine into batrachotoxins of the poison-dart frog Phyllobates aurotaenia or into the bicyclic alkaloids ofDendrobatespumilio after 6 weeks of intraperitoneal injections (27). Batrachotoxins have been detected from amphibians only in the five species of Phyllobates, the true poison-dart frogs, which were defined as a monophyletic group, partly in recognition of the occurrence of batrachotoxins in their skin (28). Batrachotoxins are virtually absent in other tissues of these frogs. Only the three Colombian species of Phyllobates have high enough levels of batrachotoxins in their skin to make them useful for poisoning blow darts. The highest levels occur in Phyllobates terribilis, which has roughly 500 pg batrachotoxin, 300 p g homobatrachotoxin, and 200 pg batrachotoxinin A per frog skin ( 1 ) . This frog is so toxic that Indians merely scrape the grooved tips of blow darts across the frog’s back (28). With the less toxic Phyllobates bicolor and Phyllobates aurotaenia, both of which contain roughly 20 pg batrachotoxin, 10 p g homobatrachotoxin, and 50 p g batrachotoxinin A per frog skin (I), the frog is impaled on a stick in order to provoke a profuse skin secretion containing the batrachotoxins that is used to poison the blow darts. The remaining two species of Phyllobates, both Central American species, have very low levels of batrachotoxins. The Costa Rican species, Phylfobates vittatus, has only 0.2 p g batrachotoxin, 0.2 pg homobatrachotoxin, and 2 pg batrachotoxinin A per frog skin ( 1 ) . Several populations of the Panamanian-Costa Rican species Phyllobates lugubris do not have levels of batrachotoxins sufficient to be detectable either by toxicity or by the sensitive Ehrlich color reaction (Ref. 1 and J. W. Daly, unpublished data). One Panamanian population does contain very low levels (estimated) of 0.2 p g batrachotoxin, 0.1 pg homobatrachotoxin, and 0.5 p g batrachotoxinin A per frog skin (1). Frogs of the genus Phylfobates contain other classes of alkaloids. Usually these are present only in trace amounts, although the montane Colombian species Phyllobates bicolor contains significant amounts (100 p g or more per frog skin) of decahydroquinolines and histrionicotoxins (I). In view of the apparently extremely limited occurrence of batrachotoxins in Nature, namely, in the five monophyletic species of the dendrobatid

3.

AMPHIBIAN ALKALOIDS

193

genus Phyllobates, and their absence in other dendrobatid species and in many other species of amphibians examined for lipophilic alkaloids (see Ref. I ) , it seems likely that a complete set of biosynthetic enzymes responsible for formation of batrachotoxins has evolved in this monophyletic group of dendrobatid frogs. A dietary source seems unlikely. However, levels of batrachotoxin/homobatrachotoxinin Phyllobates terribilis did decline slowly when wild-caught frogs were maintained in terraria on fruit flies and crickets (29). After 3 years, levels were 320 and 480 pg in two frogs. After 6 years, levels were 250 p g in one frog, but were 1150 p g in another frog sacrificed because of bloating. Wild-caught frogs had levels of 1140 ? 140 p g ( n = 10). Second generation terrarium-reared Phyllobates terribilis did not have detectable levels of batrachotoxins; that is, if present at all, levels of batrachotoxins were more than 10,000-fold lower than in the wild-caught parents (29). Thus, unresolved questions concerning the synthesis of batrachotoxins by the poison-dart frogs are raised. Are they present in the food chain? If so, how are they concentrated selectively by Phyllobates terribilis > P . aurotaenia and P . bicolor % P . vittatus > P . lugubris, and not by other dendrobatid frogs? The diet of Phyllobates and other dendrobatid frogs consists entirely of small insects, such as ants, fruit flies, mosquitoes, crickets, and termites. Is there an essential cofactor or precursor present in the diet of wild Phyllobates, but not in the fruit fly and cricket diet of frogs maintained in terraria? If so, it seems likely not to be from the wild insects per s e , but more likely to be from the gut content of the insects. Or is there a symbiotic organism present in the wild frogs that is needed for elaboration of batrachotoxins? Or is there an environmental trigger that turns on and/or maintains expression of frog enzymes responsible for synthesis of batrachotoxins? It is noteworthy that P . aurotaenia and P . terribilis, both wild-caught and captive-raised, have voltage-dependent sodium channels that are insensitive to batrachotoxin (29,30).Thus, such frogs have evolved a batrachotoxin-resistant sodium channel. Other dendrobatid frogs, for example, Dendrobates histrionicus, have sodium channels sensitive to batrachotoxin (cited in Ref. 29). The same questions concerning the origin of batrachotoxins in true poison-dart frogs pertain to other dendrobatid alkaloids, since dendrobatid poison frogs of the genera Dendrobates and Epipedobates also do not contain alkaloids when reared in captivity (31). One of the batrachotoxin alkaloids has now been discovered in a nonamphibian source. Remarkably, the source is the skin and feathers of New Guinea birds of the genus Pitohui (family Pachycephallinae) (32). Of the three species examined, highest levels occurred in the hooded pitohui (Pitohui dichrous), where it is estimated that the skin and feathers of one bird contained about 20 p g of homobatrachotoxin. Batrachotoxin was not

194


detected. Striated muscles contained much lower levels of homobatrachotoxin. The bird is recognized by natives of New Guinea as being toxic, and they advise that it should be skinned prior to cooking if it is eaten. This discovery of homobatrachotoxin in a bird suggests the independent development of biosynthetic pathways leading to batrachotoxins in birds (Pitohui) and frogs (Phylfobates).Birds of the genus Pitohui subsist on insects and seeds. None of the frogs of New Guinea are from a genus that produces alkaloids. The levels of batrachotoxins in the skin of Phyllobates terribilis (-2000 pg/g skin) are orders of magnitude higher than the levels of hombatrachotoxin in the skin of the hooded pitohui (-5 pg/g skin).

B. SAMANDARINES

I . Structures The isolation and characterization of samandarine and its congeners from the parotoid skin glands of the European fire salamander (Salamandra salamandra) and alpine salamander (Salamandra atra) have been reviewed in detail (8-lo), most recently in 1986 (5).The majority of isolations have been from two subspecies of Salamandra salamandra. The 1986 review also covers synthetic approaches to the samandarines. Samandarine (11) proved to be a steroidal alkaloid containing an oxazolidine in an altered A ring. The ring junctions are as in cholestane. Since these studies were essentially completed prior to the emergence of mass spectrometry and nuclear magnetic resonance spectroscopy, structure elucidation relied mainly on chemical conversions, infrared and ultraviolet spectroscopy, and X-ray crystallography. The majority of the samandarine alkaloids contained the oxazolidine ring (samandarine, 0-acetylsamandarine, sa-

19

OR

Samandarine (11)

3. AMPHIBIAN

195

ALKALOIDS

mandarone, samandaridine, samandenone, samandinine), which affords a diagnostic pair of infrared absorbances in the region from 830 to 875 cm-' (33). The salts of the samandarines show only a weak absorbance at 870 cm-' . Structures of the nine naturally occurring samandarine alkaloids are shown in Fig. 2. Cycloneosamandione did not contain the oxazolidine ring, but rather a tautomeric aminocarbonyl carbinolamine system (34). Two structures were proposed, one a carbinolamine formed with a C-19 aldehyde, the other a carbinolamineformed with a 6-keto group. Ultimately, the cyclone-

*

dO H0 Samandarine 0-Acety lsamandarine

Samandarone

R=H R = COCH3

HN O,

HN ,.@

i

yp \

Samandaridine

Samandinine

HN/A O, :

@o N

fl0 Samandenone

HN&OH

-manine

Cycloneosamandione N "1'.

I......,

... 6

k

OH

lsocycloneosamandaridine FIG.2. Structures of samandarine alkaloids isolated from salamanders of the genus Salamandra.

196

JOHN W. DALY E T A L .

osamandione was demonstrated to have the C-19 carbinolamine structure shown in Fig. 2 (35;see also Ref. 5 ) . One other samandarine alkaloid, namely cycloneosamandaridine, contained a tautomeric aminocarbonyl e carbinolamine system, and it was initially thought to be related to samandaridine just as cycloneosamandione was related to samandarone (34). However, synthetic cycloneosamandaridine with the carbinolamine formed with a C- 19 aldehyde was not identical with the natural compound (36), which thus appears to have a carbinolamine formed with a 6-keto group. Retention of the original name, cycloneosamandaridine, for the natural compound would be unfortunate, since it implies the presence of the cycloneosamandione A,B ring system. The name isocycloneosamandaridine, proposed in Ref. 5 , is used in this chapter (see Fig. 2). The name cycloneosamandaridine was retained for the synthetic material, which is shown in Fig. 3; it has not been reported in Nature. There is one other natural samandarine alkaloid that does not contain the oxazolidine ring, but instead a seven-membered nitrogen-containing A ring. The structure of this alkaloid, samanine, is shown in Fig. 2. A synthetic alkaloid, isomeric with samandarine, has been erroneously reported to be isolated from a natural source, namely, the salamander Cryprobrunchus muximus (37). However, S. Hara has informed us that this alkaloid was never isolated from Nature (see discussion in Ref. 5). It is referred to in Fig. 3 as the Hara-Oka alkaloid. Physical and spectral properties of samandarine alkaloids are presented in Table 11. Mass spectra of various samandarine alkaloids and derivatives have been presented (38-43). Fragments of C,H,NO+ (mlz 86) and C,H,NO ( m / z 85) are typical of oxazolidine-containing samandarine alkaloids. Infrared spectra of various samandarine alkaloids have been published (33,34,43-46 and references therein). Proton magnetic resonance spectra for samandarone, samandenone, and cycloneosamandione have been presented (38,40,41). Samandarine, samandarone, and +

Cycloneosamandaridine "Hara-Oka alkaloid" FIG. 3. Synthetic sarnandarine alkaloids. These compounds have not been detected in Nature.

3.

AMPHIBIAN ALKALOIDS

I97

TABLE I1 PHYSICAL A N D SPECTRAL PROPERTIES OF SAMANDARINE ALKALOIDS (33-36.38-47) Samandarine, C IN02 Mass spectrum: rnlz 305(35), 277(14), 86(94), 85(100). 57(36), 56(50) Infrared: 853,832 cm-' Optical rotation: [a];+ 29.5" mp (free base): 188°C Rr value: 0.34" 0-Acetylsamandarine, C21H31N0, Mass spectrum: unpublished Infrared: 1730, 1240,840, 830 cm-' mp (free base): 158- 159°C Rr value; 0.49 Samandarone, C 19H29N02 Mass spectrum: rnlz 303(27), 275(12), 86(24), 85(lOO), 57(32), 56(66) Infrared: 1740,845, 831 cm-' Optical rotation: [a];- 115.7" mp (free base): 190°C Samandaridine, CZlHI I NO, Mass spectrum: rnlz 34326). 317(13), 86(17),85(100), 57(33), 56(44) Infrared: 1760, 840, 830 cm-' Optical rotation: [a]:;,+ , 14.1" mp (free base): 287-288°C Rr value: 0.40 Samandenone, C22H,lN02 Mass spectrum: mlz 343(100),328(18),31320). 300(8), 283(1 I ) , 259(13), 245(10), 148(17), 86(85), 85(93), 57(67),56(70) Infrared: 1678, 1611, 845, 828 cm-1 mp (free base): 189-191°C Samandinine, C24H19N01 Mass spectrum: rnlz 389, 85,57, 56 Infrared: 1725, 1240, 845. 820 cm-' mp: 170°C Cycloneosamandione, C19H?9N02 Mass spectrum: rnlz 303(l8), 275(10), 274(26), 57(48), 56(35) Infrared: 1750 cm-' mp (free base): 118-1 19°C Rr value: 0.43 (conrinued)

198

JOHN

w. DALY

E ~ A L .

TABLE I1 (Continued) Isocycloneosamandaridine,bC21H,IN03 Mass spectrum: mlz 345(3), 344(14), 330(53), 97(26), 9315). 85(32), 83(30), 71(41), 69(55), 57(55), 56(100) Infrared: 1780 cm-I mp (free base): 28 1-282°C Samanine, C19H3,N0 Mass spectrum: mlz 291(100), 276(71), 96(38), 82(22), 70(28), 57(70), 56(90) Infrared (N.0-diacetyl): 1735, 1640 cm-I mp (free base): 193-195°C "Thin-layer chromatography: silica gel, CHC1,-MeOH (9:I , vlv). Formerly referred to as cycloneosamandaridine (43).

0-acetylsamandarine elute at 220, 223, and 243"C, respectively, on the temperature-programmed, packed OV- 1 columns used for the analysis of dendrobatid alkaloids (see Appendix).

2 . Biological Activity Samandarine is a relatively potent, centrally active neurotoxin with an injected lethal dose for a mouse of about 70 pg (see Ref. 5 , and references therein). Samandarone is somewhat less toxic. Convulsions, respiratory failure, cardiac arrhythmias, and partial paralysis precede death. The fire salamander is sensitive to its own toxin. There have been no recent studies on the pharmacology of samandarine alkaloids. Samandarine is a potent local anesthetic (48). Cardiac depressant effects (48,49)and inhibition of binding of a radiolabeled batrachotoxin analog to sodium channels (50) are consonant with the potent local anesthetic activity of samandarine. Samandarine alkaloids show antimicrobial activity (51 and references therein). 3. Occurrence

The European fire salamander (Salamandra salamandra) and the alpine salamander (Salamandra atra) are the only amphibians known to contain samandarine alkaloids. These are the only two species in this genus. The proposal that extracts of the brilliant black and yellow Australian myobatrachid frog Pseudophryne corroboree contained samandarine alkaloids (52) has proved to be incorrect, and this and other frogs of the genus Pseudophryne instead contain pumiliotoxins and pseudophrynamines (see Sections III,C and VI1,A). The major alkaloids of Salamandra salamandra are samandarine, samandarone, and 0-acetylsamandarine. There do not

3.

AMPHIBIAN ALKALOIDS

199

appear to be any alkaloids related in structure to samandarines found elsewhere in Nature. In contrast to dendrobatid alkaloids, which are not present in captiveraised dendrobatid frogs, fire salamanders produce samandarine alkaloids when reared in captivity (G. Habermehl, personal communication, 1989). No apparent differences in alkaloid content occurred for at least three generations. Incubation of secretions from salamander parotoid glands with radiolabeled cholesterol in buffer for 3 days at room temperature led to some apparent incorporation of radioactivity into samandarine alkaloids

(53). 111. Bicyclic Alkaloids Most amphibian alkaloids are not as complex in structure as the steroidal batrachotoxins and samandarines. Of the 300 known amphibian alkaloids, most have been characterized from the skin extracts of frogs of the family Dendrobatidae and, hence, have been referred to as dendrobatid alkaloids. The major bicyclic classes of dendrobatid alkaloids are the histrionicotoxins, decahydroquinolines, and pumiliotoxin-A class. Because of the presence of a piperidine ring in most dendrobatid alkaloids, they also have been referred to as piperidine-based alkaloids. It was apparent by the late 1970s that hundreds of different alkaloids would be detected in skin extracts of dendrobatid frogs, and, therefore, a code system was developed using the nominal molecular weight of the alkaloid with an added letter to distinguish alkaloids with the same nominal molecular weight. Thus, the first alkaloid reported of a particular molecular weight had no attached letter, but later addition of an A was required for this alkaloid when another alkaloid with the same nominal molecular weight was discovered (e.g., 219 became 219A, 252 became 252A). In some cases it appeared initially that two alkaloids were present as an inseparable mixture, for example, a mixture of 223A and 223B; however, several were later shown to be a single compound, and a combination of the original codes was retained, as in 223AB, 239AB, 239CD, and 269AB. To characterize isomers, prefixes (e.g., cis, trans, epi, iso) and primes (Ar,A”) have been introduced. In some cases, for example, 307A, two diastereomers 307A’ and 307A” have been isolated, and the name 307A is now used to indicate that the diastereomeric composition of the alkaloid was, or is, unknown. Certain of the dendrobatid alkaloids also have been given trivial names, as in the various histrionicotoxins, pumiliotoxin C (now referred to as the decahydroquinoline cis-195A), pumiliotoxins A and B, gephyrotoxin, and epibatidine. Structure elucidation of major

JOHN W.DALY ET AL.

200

alkaloids has been mainly by X-ray crystallography and nuclear magnetic resonance spectroscopy, while minor and trace components have been characterized by gas chromatography-mass spectrometry, gas chromatography-Fourier transform infrared (FTIR) spectroscopy, and microchemical manipulations (see Appendix). A. HISTRIONICOTOXINS (AZASPIRO[5.5]UNDECANOLS)

I . Structures The structures of histrionicotoxin (283A)and dihydroisohistrionicotoxin (285A)were determined by X-ray crystallography (54) to be as shown in 111 and IV. Structures of most of the other histrionicotoxins have been

Histrionicotoxin 283A (111)

Dihydroisohistrionicotoxin 285A

(1V)

3.

20 1

AMPHIBIAN ALKALOIDS

based on nuclear magnetic resonance spectral analysis. At present, sixteen histrionicotoxins have been detected (I ,55). Nine have unsaturated pentyl (2 position) and butyl (7 position) side chains, while another seven have instead a three-carbon side chain at the 2 position. Three of the latter group have a two-carbon side chain at the 7 position. Structures of the known histrionicotoxins are shown in Fig. 4. Mass spectra of histrionicotoxins show a characteristic pathway for loss of the 2-substituent and a characteristic fragment ion at mlz 96 (C,HIoN+).Mass spectral fragmentation pathways for histrionicotoxins have been discussed (55,57; see also tabulations in Ref. 3 and data in Refs. 54,58,59). The properties of the natural histrionicotoxins are presented below in a format introduced in 1978 (60) for the dendrobatid alkaloids. The entries are as follows: (1) the code designation based on molecular weight and identifying letter(s) in boldface; (2) the trivial name, if any; (3) an empirical formula based on high-resolution mass spectrometry (tentative formulas

OH

R'

15-Carbon H i s t r i o n i c o t o x i n s 235A 237F 239H

-CH,CH=CH, -CH,CH,CH, -CH,CH,CH,

-CH=CH, -CH=CH, -CH,CH,

17-Carbon H i s t r i o n i c o t o x i n s 259A 261 263C 265E

-CH,CH=CH, -CH2CH=CH2 -CH,CH=CH, -CH,CH,CH,

cis-CH=CHC-CH cis-CH=CHCH=CH, -CH,CH,CH=CH, -CH2CH2CH=CH2

19-Carbon H i s t r i o n i c o t o x i n s 283A 285A 2858 285C 285E 287A 2878 287D 291A

c i s-CH,CH=CHC=CH -CH,CH CH-C-CH, c i~ - C & C H ~ ~ C = C H -CH2CH CH C=CH c i s-C&Ctf=cHCH=CH, -CH,CH CH=C=CH c i s-C&CH=CHCtf=CH, -CH,CH,CH,C=CH -CH2CH2CH2CH=CH2

cis-CH=CHC=CH cis-CH=CHC-CH cis-CH=CHCH=CH, cis-CH=CHCsCH cis-CH=CHC-CH cis-CH=CHCH=CH, cis-CH=CHCH=CH, cis-CH=CHCH=CH, -CH,CH,CH=CH,

FIG.4. S t r u c t u r e s of h i s t r i o n i c o t o x i n s from dendrobatid frogs.

202

JOHN W. DALY E T A L .

are indicated by single quotes); (4) an &value (silica gel, CHC1,-CH,OH, 9:l,v/v); (5) the emergent temperature on a 1.5% 6-ft OV-I-packed gas chromatographic column programmed from 150 to 280°C at 10°C per minute; (6) the electron impact-mass spectral ions followed in parentheses with intensities relative to the base peak set equal to 100, although not all peaks, in particular low-mass (rnlz < 60) and low-intensity peaks, are reported (in some cases pseudo-electron impact spectra obtained with an ion trap instrument are reported); (7) the number of hydrogens exchangeable with deuteroammonia (OD, ID, etc., meaning no exchangeable, one exchangeable, etc.); (8) citations to vapor phase-FTIR spectra or data (infrared data for solutions are so indicated); (9) perhydrogenation derivative (H,, no addition of hydrogen; H2, addition of two hydrogens, etc.); and (10) pertinent comments. Omissions indicate that no or only ambiguous data are available (for further details, see Appendix). Histrionicotoxins

235A. CI5H2,NO, 0.36, 176"C, rnlz 235(5), 234(2), 218( 1 9 , 194(76), 176(25), 150(8), 96(100). 2D. Infrared spectrum (55). H, derivative, rnlz 239, 196, 178, 96. 237F.CI5H2,NO,-, 180"C, rnlz 237(5), 220(3), 194(54),96( 100). 2D. H, derivative. 2398.C15H2,N0,-, 182"C, rnlz 239(7), 238(4), 221(6), 196(35), 178(lo), 96( 100). 2D. H, derivative. 259A. CI7Hz5NO,0.36, 190"C, rnlz 259(4), 242(2), 218(18), 200(6), 96( 100). 2D. Infrared spectrum (55). H, derivative, rnlz 267(20), 250( 13), 224(39), 196(15), 168(19), 152(100), 96(68). 261. C17H2,N0,-, 19OoC, rnlz 261(8), 220(100), 204(10), 96(68). 2D. Infrared spectrum (55).H, derivative. 263C.C17H,,N0, -, 192"C, mlz 263(1), 222(100), 204(10), 96(48). 2D, H, derivative. 2653.C,,H,,NO, -, 194"C, rnlz 265(5),264(3),248( lo), 224(48), 222(20), 152(loo), 139(63),96(95). 2D. Infrared spectrum (55). H, derivative. 283A. Histrionicotoxin, CI9Hz5NO,0.50, 210"C, rnlz 283(9), 282(2), 266(5), 218(48), 200(27), 160(22), 96( 100). 2D. Infrared spectrum (55). H,, derivative, 0.36, 214"C, m l z 2 9 3 12), 294(2), 278( 131, 252( 18), 224(73), 196(27), 180(IOO), 168(39), 96(68). A A-17-trans-histrionicotoxin (283A') can occur in trace amounts with 283A but may be an artifact (55). 285A.Isodihydrohistrionicotoxin, C,,H,,NO, 0.39, 215"C, rnlz 285(7), 284(2), 268(8), 252(12), 238(3), 218(6), 200(9), 176(24), 162(18),96(100).2D. Infrared spectrum (55). HI, derivative.

3. AMPHIBIAN

ALKALOIDS

203

285B.Neodihydrohistrionicotoxin, C,,H,,NO, 0.46, 21 l"C, mlz 285(4), 284( l), 268(3), 220(37), 202(9), 160(20), 96( 100). 2D. Infrared spectrum (55). HI, derivative. 285C. Allodihydrohistrionicotoxin, C,,H,,NO, 0.40, 21 1"C, mlz 285(4), 284(1), 268(2), 218(5), 176(15), 162(17), 96(100). 2D. Infrared spectrum (55). HI, derivative. 285E. Dihydrohistrionicotoxin, CI9H2,NO, 0.50,212"C, mlz 285( 13), 268( lo), 218( loo), 200(84), 176(13), 96(78). 2D. Infrared spectrum (55). HI, derivative. 287A. Isotetrahydrohistrionicotoxin, C,,H,,NO, 0.42, 216"C, mlz 287( 12), 286(4), 270(3), 220(30), 202(34), 176(45), 162(60), 148(24),96( 100). 2D. Infrared spectrum (55) . H, derivative. 287B.Tetrahydrohistrionicotoxin, C,,H,,NO, 0.43,213"C, mlz 287( 13), 286(2), 270(2), 220(43), 202(18), 96( 100). 2D. Infrared spectrum (55). H, derivative. 287D. Allotetrahydrohistrionicotoxin, CI9H,,NO, 0.35. 215"C, mlz 287( 14), 270(8), 220(24), 202(36), 176(38), 162(49), 96( 100). 2D. H, derivative. 291A.Octahydrohistrionicotoxin, C,,H,,NO, 0.35, 212"C, mlz 291( 12), 290(2), 274(14), 250(54), 222(24), 194(18), 192(12), 178(loo), 96(52). 2D. Infrared spectrum (55). H, derivative. Other physical and spectral properties of the histrionicotoxins are presented in Table 111. Proton and carbon-13 magnetic resonance assignments have been presented (57-59) and reviewed (3). For proton spectra, see Refs. 3,57-59; for carbon-13 assignments, see Ref. 55. The diagnostic infrared peaks for various histrionicotoxins in solution have been tabulated ( 3 3 . Vapor-phase FTIR spectra of histrionicotoxins and derivatives have been presented and discussed (55). The pK, values for histrionicotoxin and its reduction product, perhydrohistrionicotoxin,were in the range 9.0-9.3 as determined by titration. Butylboronic acid derivatives of histrionicotoxins can be easily prepared and provide several advantages for gas chromatographic-mass spectral and FTIR analysis (55). The cis-diene-containing histrionicotoxins (e.g., 285B, 2853, 287A, 287B) are slowly isomerized to a photostationary state containing 55% truns-dienes by ambient light (55). The cis-enyne moiety, although not so easily photoisomerized, also undergoes isomerization to truns-enynes, making it likely that 283A', a previously isolated 7-(truns-buten-3-yne) isomer of histrionicotoxin (59), is an artifact. Other possible artifacts are two formaldehyde condensation products (oxazolidines) of molecular weights 297 and 299 from 285A (or 285C) and 287A,respectively, detected in some extracts and evidently arising from traces of formaldehyde in the methanol used for extraction (55).

204


TABLE 111 PHYSICAL A N D SPECTRAL PROPERTIES OF HISTRIONICOTOXINS (535) 235A Optical rotation: [ale - 38.6"' (1.75, CHCI,) Histrionicotoxin (283A) mp (free base): 79-80°C Ultraviolet: A,, 224 nm, E 22,300 (C2H50H) Optical rotation: [a];5- 96.30b (HCI, I .O, C2H50H)

Isodihydrohistrionicotoxin (USA) Ultraviolet: A,,, 225 nm, E 8100, 235 nm, E 7200 (C2H50H) Optical rotation: [a]$-35.3" (HCI, 0.5, C2H50H) Neodihydrohistrionicotoxin (285B) Ultraviolet: A,,, 225 nm, E 17,300 (C2H50H) Optical rotation: [a];' - 125.9" (HCI, 1.1, C2H50H)

Allodihydrohistrionicotoxin (285C) Optical rotation: [a];' -43.4" (HCI, 1.2, C2H50H) Dihydrohistrionicotoxin (285E) Ultraviolet: A,,, 226 nm, E 24,700 (C2H50H) Optical rotation: [aid5- 122" (HCI, 1.0. C2H50H)

Isotetrahydrohistrionicotoxin (287A) E 19,200 (C2H50H)

Ultraviolet; A,,

228 nm,

Ultraviolet: A,,,

228 nm,

Tetrahydrohistrionicotoxin (287B) E 3900 (C2H50H)

Perhydrohistrionicotoxin (synthetic reduction product of 19-carbon histrionicotoxins) Optical rotation: [a]$- 34.6", -36.2" (HCI, 1.0, C2H50H,CHCI,) "Synthetic 235A had an [a]%of bSynthelic 283A had an [a]?of

-

102" (1.82. C2HcOH)(56). 114" (1.06, C,H,OH) (56).

2. Biological Activity

Histrionicotoxins have relatively low toxicities, and thus the toxin designation is a misnomer. A subcutaneous dose of 1000 p g of either histrionicotoxin o r isodihydrohistrionicotoxin in a mouse causes locomotor difficulties and prostration (54,60). Pharmacologically, the histrionicotoxins affect at least three classes of channels in nerve and muscle. The first class of channels are the receptorregulated channels, in particular the nicotinic acetylcholine receptorchannel, where histrionicotoxins, in a time- and stimulus-dependent man-

3.

AMPHIBIAN ALKALOIDS

205

ner, block the channel conductance and accelerate the desensitization or inactivation of the channel. The effects are those of a so-called noncompetitive blocker, and indeed the histrionicotoxins now represent classic noncompetitive blockers for nicotinic receptor-channel complexes (61-64). The development of a [3H]perhydro derivative of histrionicotoxin has afforded a radioligand for investigation of noncompetitive binding sites on the nicotinic receptor-channel complex. Histrionicotoxins also block conductance of a receptor-regulated channel that is activated by glutamate or N-methylaspartate (65,66). The second class of channels are the voltage-dependent sodium channels, where histrionicotoxins reduce conductances in a manner reminiscent of local anesthetics (61). The third class are the voltage-dependent potassium channels, where histrionicotoxins reduce conductances in a time- and stimulus-dependent manner. Structure-activity relationships for histrionicotoxins differ at the three classes of channels (61). A summary and overview of the extensive studies of the biological effects of histrionicotoxins appeared in 1986 (5). Most of the studies have focused on cholinergic systems. Since that time, many articles have appeared. It is beyond the scope of the present review to do more than continues highlight selected developments. [3H]Perhydr~histrionicotoxin to be used as a probe for noncompetitive blocker sites on the muscle-type nicotinic receptor-channels of the electric ray electroplax (64). Recent studies on ganglion (67), central neuronal (63), pheochromocytoma ( 6 4 , and adrenal chromaffin cells (68) demonstrate that histrionicotoxins are not only potent noncompetitive blockers of muscle-type nicotinic receptor-channels, but also noncompetitive blockers of ganglionic-type and central neuronal-type nicotinic receptor-channels. Histrionicotoxin blocks neuromuscular transmission in preparations of Dendrobates histrionicus, but higher concentrations are required to cause blockade than in the ranid frog Rana pipiens (69). 3. Occurrence

Histrionicotoxins represent a unique structural class of alkaloids found only in dendrobatid frogs (see below). Somewhat similar hydroxyazaspiro-undecanes, namely, sibirine, nitramine, and isonitramine, occur in certain plants of the genus Nitraria (cf. Ref. 70). Histrionicotoxins were first discovered in the extremely variable dendrobatid species Dendrobates histrionicus of western Colombia and northwestern Ecuador (54). Histrionicotoxin (238A),allodihydrohistrionicotoxin (285C), and isodihydrohistrionicotoxin (285A) represent major alkaloids in nearly all populations of this species (1,71). In one population from northwestern Ecuador, octahydrohistrionicotoxin (291A)is a major alkaloid (71), whereas in one population from the lower Rio San Juan

206

JOHN W . D A L Y E T A L .

drainage of western Colombia the lower molecular weight histrionicotoxins 235A and 259A have replaced the 19-carbon 283A, 285C, and 285A as major alkaloids ( J. W. Daly, unpublished). No histrionicotoxins are present in the “sibling” Colombian species Dendrobutes lehmanni. Histrionicotoxins are present in skin extracts from many dendrobatid frogs, representing major alkaloids in Dendrobates auratus, D . azureus, D . granuliferus, D . histrionicus, D . leucomelus, D . occultator, D . pumilio (some, but not all populations), D . quinqueuittatus, D . reticulatus, D . tinctorius, D . truncatus, and D . uentrimaculatirs (I). Histrionicotoxins are absent or trace alkaloids in D . arboreus, D . new species (Panama), D. lehmanni, D . pumilio (some populations), and D . speciosirs. In the dendrobatid genus Epipedobates, histrionicotoxins are major alkaloids in E. espinosai, E . paruulus, E . petersi, E . pictus, and E . triuittatirs, but are absent or trace alkaloids in the six other species examined (I). In the dendrobatid genus Minyobates, histrionicotoxins are absent in the nine species that have been examined (I). In the dendrobatid genus Phyllobates, a genus that is typified by the presence of batrachotoxins, histrionicotoxins occur as major alkaloids in P . bicolor, but are trace alkaloids or absent in the remaining four species (I). The distribution of histrionicotoxins within species and genera of dendrobatid frogs argues strongly for genetic determinants for their occurrence. Histrionicotoxins are often accompanied by highly unsaturated decahydroquinolines in dendrobatid frogs. N o deoxyhistrionicotoxins have yet been detected. In 1984, dendrobatid alkaloids were reported from one genus of each of three other amphibian families, namely, Bufonidae, Myobatrachidae, and Ranidae (72). Histrionicotoxin (283A), allodihydrohistrionicotoxin (285C),and isodihydrohistrionicotoxin (285A) were present as major alkaloids in a single skin of the Madagascan ranid frog Mantella madagascariensis obtained from a commercial dealer (72). Subsequent fieldcollected specimens of Mantella mudagascariensis have contained a variety of alkaloids, but no histrionicotoxins (73). Thus, histrionicotoxins may prove to be unique to certain dendrobatid species of the genera Dendrobates, Epipedobates, and Phyllobates.

B. DECAHYDROQUINOLINES I . Structures The first decahydroquinoline found in amphibians was isolated, along with two other alkaloids, from skin extracts of a Panamanian dendrobatid frog, Dendrobates pumilio. The three alkaloids were designated pumiliotoxins A, B, and C (74,75). Pumiliotoxins A and B were quite toxic, and

3. AMPHIBIAN

ALKALOIDS

207

are the parent members of the pumiliotoxin-A class of alkaloids (Section 111,C). Pumiliotoxin C proved to be a decahydroquinoline, namely, (2S,4aS,5R,8aR)-5-methyl-2-n-propyl-cis-decahydroquinoline, as shown in V. It was relatively nontoxic, and hence the toxin designation is inappropriate; the nomenclature decahydroquinoline cis-l95A, used in recent publications and in this chapter, is much preferable, particularly to avoid confusion with the structurally dissimilar pumiliotoxin-A class. A range of decahydroquinolines have been isolated from skin extracts of dendrobatid frogs. Structures of several have been determined (76-78) through the analysis of mass spectra, nuclear magnetic resonance spectra, and FTIR spectra. Both cis- and trans-fused decahydroquinolines have been isolated. The decahydroquinolines show a major mass spectral fragment ion corresponding to loss of the 2-substituent and a minor fragment ion from loss of the 5-substituent. Although the mass spectra of cis- and trans-isomers are virtually identical, the FTIR spectra are not and appear to be diagnostic for the ring-fusion stereochemistry (78). Thus, transdecahydroquinolines, such as trans-219A, have sharp, single peaks at approximately 1 100 and 1300 cm-', whereas cis-fused decahydroquinolines, such as cis-195A and cis-219A, show doublets at around 1120 and 1340 cm-' (78). The doublets were proposed to arise from contributions from two cis-fused conformations (78). Several diastereomers of cis-195A have been synthesized, namely, the 2-epi (78a),the 5-epi and 8a-epi (79), and the 2-epi-8a-epi analogs (80). Structures of 15 of the relatively wellcharacterized decahydroquinolines are shown in Fig. 5 . A number of other dendrobatid alkaloids appear to be decahydroquinolines based on spectral and chemical properties. All of these alkaloids are secondary amines; no simple N-alkyl derivatives have been found. FTIR spectra are not yet available for many of these alkaloids. Many decahydro-

Decahydroquinoline cis-195A Purniliotoxin C

(V)

208


br, /J 8a N

.b 2

H o ~ . . \ " ,

H

H

I H

H

cis-195A

cis-21 1 A

cis-219A

cis-223F

I

H

H

H

cis-249D

cis-243A

cis-275B

A-

r

&l OJ J0 J T C ) J l- i N l

H I

H

trans-1 95A

H I

H

trans-219A

trans-223F

H I

H

trans-243A

lh/b&elH0& H I NI

5-epi-trans-243A

H HI

trans-249D

l iH l

trans-249E

l iH l

trans-253D

FIG.5 . Structures of decahydroquinolines from amphibians. Absolute configurations of natural cis-195A and rrans-219A are known. Only the relative configurations are known for the others.

quinolines show a major fragment ion corresponding to Cl,H18N+ (mlz 152), as in the case of cis-195A. These would have a methyl group as a 5-substituent. One alkaloid (189)that appears to be a tetrahydroquinoline and one (193D) that appears to be an octahydroquinoline have been reported from ranid frogs of the genus Manteffa(73). The decahydroquinoline alkaloids are tabulated below, along with the tetrahydroquinoline and the octahydroquinoline. Tentative structures are suggested for several alkaloids that are not shown in Fig. 5. Decahydroquinolines 153A. 'C,oH,9N,'--,1540C, mlz 153( loo), 152(60). ID. H, derivative. Tentative structure: a 5-methyldecahydroquinoline.


209

167D. ‘Cl,H2,N,’-, 154”C, mlz 167(100), 166(53). ID. H, derivative. Tentative structure: a 5-ethyldecahydroquinoline. 181D. ‘C,2H23N,’--, 156”C, mlz 181(3), 152(100). ID. Tentative structure: a 2-ethyl-5-methyldecahydroquinoline. 181E. ‘CI2H,,N,’--, 156”C, mlz l8l( loo), 180(46). ID. H, derivative. Tentative structure: a 5-propyldecahydroquinoline. 189. ‘C13H19N,’-,-, ion trap, mlz 190(10), 174(26), l6l( IOO), 146(94), 91( 10). OD. Infrared data (73). Tentative structure: a 2-propyl-5-methyl-

5,6,7,8-tetrahydroquinoline. 193D. ‘C13H23N,’-,-, ion trap, mlz 193(5), l50( IOO), 122(12), 96( 12). ID. Infrared data (73).Tentative structure: a 2-propyl-5-methyl octahydroquinoline. 195A. Pumiliotoxin C, C,,H,,N, 0.20, 157”C,mlz 195(3), 194(5), 180(1), 152(100), 109(8). ID. Infrared spectrum (78), infrared data (73). H, derivative. N-Acetyl derivative. Both cis and trans isomers occur, with pumiliotoxin C being the cis isomer. 211A. C,,H,,NO,--, 166”C, mlz 21 l(3), 210(2), 168(100), 152(32), 150(13). 2D. H, derivative. Only a cis isomer has been detected (77). 219A. C,,H,,N, 0.32, 165°C mlz 219( I), 218(2), 178(100). ID. H,derivative, mlz 223, 180. Infrared spectra (78). N-Acetyl derivative. Two naturally occurring isomers, cis-219A and truns-219A, do not separate on packed columns. Another isomer 219A‘ emerging at 164°C may represent a 2-epi-cis-219A. 219C. C,,H,,N,-, 170”C, mlz 219(

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